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Author(s): Shu-Zhen Wang
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METHODS IN MOLECULAR BIOLOGY™
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Retinal Development
Methods and Protocols
Edited by
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Editor
Shu-Zhen Wang
Department of Ophthalmology
University of Alabama at Birmingham
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ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-61779-847-4 ISBN 978-1-61779-848-1 (eBook)
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Preface
In recent years, there have been major advances in the concepts and methodologies used in
the study of retinal development at both cellular and molecular levels. These advanced
methodologies have allowed and will continue to allow researchers to gain new insights
into the molecular mechanisms underlying retinal development. Additionally, the retina,
being part of the central nervous system (CNS) and accessible for direct, in vivo experimen-
tal manipulations, has historically served and continues to serve as an “eye” to “seeing” the
molecular and cellular events underpinning CNS development.
In this volume, a group of distinguished researchers offer insightful and detailed proto-
cols for a wide range of experiments that fall into six theme categories. Theme I describes
several methodologies for manipulating gene expression in vivo, including conditional gene
inactivation (knockout), generation of transgenic Xenopus, and electroporation of embry-
onic chick and adult mouse eyes. These methodologies are instrumental in uncovering the
molecular mechanisms of retinal development, while some may be adapted to investigating
potential therapeutic reagents. Theme II focuses on techniques for tracing cell fates with
modernized classic blastomere manipulation in Xenopus and with Cre-based technique in
mouse and in zebrafish. Theme III covers several protocols of in vitro systems, which have
become increasingly popular in biological and biomedical laboratories. Theme IV presents
protocols to study retinal regeneration and stem cell-based replacement, two research areas
with heightened interests due to their therapeutic implications. Theme V centers on ERG
(function) recording and noninvasive imaging, which are likely needed for future analyses
of retinal development. Theme VI is devoted to other emerging, cutting-edge methodolo-
gies, including laser microdissection for studying miRNA and DNA methylation, 3C (chro-
mosomal conformation capture), Exome-seq, and RAN-seq. These emerging methodologies
will empower investigators in the retinal development field and other fields, such as epige-
netics and gene discovery. As such, this volume provides methodologies crucial to the suc-
cess of increasingly more complex and often challenging investigations in the fields of retinal
development and other biological and biomedical research.
As in other volumes of the “Methods in Molecular Biology” series, each chapter in this
volume contains a “Notes” section, in which expert researchers offer key insightful instruc-
tions, and yet not in excessive details, to assist users of this volume to successfully execute
specific experiments.
This volume is designed as a reference manual for scientists with various levels of experi-
ence, from those who wish to set foot in the field of retinal development to those who wish
to enrich the battery of techniques used in their research. It also provides a framework of
methodologies that can be modified and applied to studies of the development of nonreti-
nal tissues, the pathological processes of certain retinal degenerative diseases, and the devel-
opment of gene- and cell-based therapies.
Birmingham, AL, USA Shu-Zhen Wang
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I MANIPULATION OF GENE EXPRESSION IN VIVO
1 Conditional Control of Gene Expression in the Mouse Retina. . . . . . . . . . . . . 3

Qian Ding and Lin Gan
2 Generation of Transgenic Xenopus Using Restriction Enzyme-Mediated
Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Mohammad Haeri and Barry E. Knox
3 In Vivo Functional Analysis of Transcription Factor: Response
Element Interaction Using Transgenic Xenopus laevis . . . . . . . . . . . . . . . . . . . 41
Heithem M. El-Hodiri, Yi Pan, and Lisa E. Kelly
4 Subretinal Delivery and Electroporation in Pigmented and Nonpigmented
Adult Mouse Eyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
John M. Nickerson, Penny Goodman, Micah A. Chrenek,
Christiana J. Bernal, Lennart Berglin, T. Michael Redmond,
and Jeffrey H. Boatright
5 In Ovo Eye Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Teri L. Belecky-Adams, Scott R. Hudson, and Sarika Tiwari
6 Targeted Microinjection of Synthetic mRNAs to Alter Retina Gene
Expression in Xenopus Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Sally A. Moody
PART II TRACING CELL FATE
7 Testing Retina Fate Commitment in Xenopus by Blastomere Deletion,

Transplantation, and Explant Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Sally A. Moody
8 Application of Cre-loxP Recombination for Lineage Tracing
of Adult Zebrafish Retinal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Rajesh Ramachandran, Aaron Reifler, Jin Wan,
and Daniel Goldman
9 Fate Tracing of neurogenin2-Expressing Cells in the Mouse Retina
Using CreER™: LacZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Wenxin Ma and Shu-Zhen Wang
vii
viii Contents
PART III IN VITRO SYSTEMS
10 In Vitro Explant Culture and Related Protocols for the Study

of Mouse Retinal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Kangxin Jin and Mengqing Xiang
11 In Vitro Biochemical Assays to Monitor Rhodopsin Function . . . . . . . . . . . . . 167
Joshua Sammons and Alecia K. Gross
12 Transfection of Primary Embryonic Chicken Retinal Cells
Using Cationic Lipid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Yi-Wen Hsieh and Xian-Jie Yang
13 Production of High-Titer RCAS Retrovirus . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Run-Tao Yan and Shu-Zhen Wang
14 Chick Retinal Pigment Epithelium Transdifferentiation Assay
for Proneural Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Shu-Zhen Wang and Run-Tao Yan
PART IV REGENERATION/STEM CELLS
15 Studying the Generation of Regenerated Retinal Neuron from Müller

Glia in the Mouse Eye. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Mike O. Karl and Thomas A. Reh
16 Production and Transplantation of Retinal Cells from Human
and Mouse Embryonic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Anna La Torre, Deepak A. Lamba, Anu Jayabalu,
and Thomas A. Reh
17 Light-Induced Photoreceptor Degeneration in the Retina
of the Zebrafish. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Scott Taylor, Jing Chen, Jing Luo, and Peter Hitchcock
18 Microarray-Based Gene Profiling Analysis of Müller Glia-Derived
Retinal Stem Cells in Light-Damaged Retinas from Adult Zebrafish . . . . . . . . 255
Zhao Qin and Pamela A. Raymond
PART V FUNCTION/IMAGING
19 Measuring Rodent Electroretinograms to Assess Retinal Function . . . . . . . . . . 265

Molly E. Clark and Timothy W. Kraft
20 Functional Imaging of Retinal Photoreceptors and Inner Neurons
Using Stimulus-Evoked Intrinsic Optical Signals . . . . . . . . . . . . . . . . . . . . . . . 277
Xin-Cheng Yao and Yi-Chao Li
Contents ix
PART VI EMERGING METHODOLOGIES
21 Use of Laser Capture Microdissection for Analysis of Retinal

mRNA/miRNA Expression and DNA Methylation . . . . . . . . . . . . . . . . . . . . . 289
Laszlo Hackler Jr, Tomohiro Masuda, Verity F. Oliver,
Shannath L. Merbs, and Donald J. Zack
22 Revealing Looping Organization of Mammalian Photoreceptor
Genes Using Chromosome Conformation Capture (3C) Assays . . . . . . . . . . . 305
Guang-Hua Peng and Shiming Chen
23 Retinal Transcriptome Profiling by Directional Next-Generation
Sequencing Using 100 ng of Total RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Matthew J. Brooks, Harsha Karur Rajasimha,
and Anand Swaroop
24 Exome Sequencing: Capture and Sequencing of All Human Coding
Regions for Disease Gene Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Rinki Ratna Priya, Harsha Karur Rajasimha,
Matthew J. Brooks, and Anand Swaroop
25 Determination of Posttranslational Modifications of Photoreceptor
Differentiation Factor NRL: Focus on SUMOylation . . . . . . . . . . . . . . . . . . . 353
Jerome E. Roger, Jacob Nellissery, and Anand Swaroop
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Contributors
TERI L. BELECKY-ADAMS • Department of Biology & Center for Regenerative

Biology and Medicine, Indiana University-Purdue University Indianapolis,
Indianapolis, IN, USA
LENNART BERGLIN • Department of Ophthalmology, Emory University,
Atlanta, GA, USA
CHRISTIANA J. BERNAL • Department of Ophthalmology, Emory University,
Atlanta, GA, USA
JEFFREY H. BOATRIGHT • Department of Ophthalmology, Emory University,
Atlanta, GA, USA
MATTHEW J. BROOKS • Neurobiology Neurodegeneration and Repair Laboratory,
National Eye Institute, National Institutes of Health, Bethesda, MD, USA
JING CHEN • Department of Ophthalmology and Visual Sciences,
University of Michigan, Ann Arbor, MI, USA
SHIMING CHEN • Department of Ophthalmology and Visual Sciences,
Washington University School of Medicine, St. Louis, MO, USA;
Department of Developmental Biology, Washington University
School of Medicine, St. Louis, MO, USA
MICAH A. CHRENEK • Department of Ophthalmology, Emory University,
Atlanta, GA, USA
MOLLY E. CLARK • Departments of Vision Sciences & Optometry,
University of Alabama at Birmingham, Birmingham, AL, USA
QIAN DING • University of Rochester Eye Institute, University of Rochester,
Rochester, NY, USA
HEITHEM M. EL-HODIRI • Center for Molecular and Human Genetics,
The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
LIN GAN • Department of Neurobiology and Anatomy, Center for Neural
Development and Disease, University of Rochester Eye Institute,
University of Rochester, Rochester, NY, USA
DANIEL GOLDMAN • Department of Biological Chemistry, Molecular & Behavioral
Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA
PENNY GOODMAN • Department of Ophthalmology, Emory University,
Atlanta, GA, USA
ALECIA K. GROSS • Department of Vision Sciences, Evelyn F. McKnight Brain
Institute University of Alabama at Birmingham, Birmingham, AL, USA;
Department of Cell Biology, University of Alabama at Birmingham,
Birmingham, AL, USA
JR. LASZLO HACKLER • Avidin Ltd, Szeged, Hungary
MOHAMMAD HAERI • Department of Neuroscience & Physiology,
SUNY Upstate Medical University, Syracuse, NY, USA
xi
xii Contributors
PETER HITCHCOCK • Department of Ophthalmology and Visual Sciences,

YI-WEN HSIEH • Department of Developmental Biology, Cincinnati Children’s
Research Foundation, Cincinnati, OH, USA
SCOTT R. HUDSON • Department of Biology & Center for Regenerative
Biology and Medicine, Indiana University-Purdue University Indianapolis,
ANU JAYABALU • Department of Biological Structure, Institute for Stem Cells
and Regenerative Medicine, University of Washington, Seattle, WA, USA
KANGXIN JIN • Department of Pediatrics, Center for Advanced Biotechnology
and Medicine, Graduate Program in Molecular Genetics, Microbiology
and Immunology, UMDNJ-Robert Wood Johnson Medical School,
Piscataway, NJ, USA
MIKE O. KARL • Department of Biological Structure, Institute for Stem Cells
LISA E. KELLY • Center for Molecular and Human Genetics, The Research
Institute at Nationwide Children’s Hospital, Columbus, OH, USA
BARRY E. KNOX • Department of Neuroscience & Physiology, SUNY Upstate
Medical University, Syracuse, NY, USA
TIMOTHY W. KRAFT • Department of Vision Sciences, University of Alabama
at Birmingham, Birmingham, AL, USA
DEEPAK A. LAMBA • Department of Biological Structure, Institute for Stem Cells
YI-CHAO LI • Department of Biomedical Engineering, University of Alabama
JING LUO • Department of Ophthalmology and Visual Sciences, University of Michigan,
Ann Arbor, MI, USA
WENXIN MA • National Eye Institute, National Institutes of Health,
Bethesda, MD, USA
TOMOHIRO MASUDA • Wilmer Eye Institute, Johns Hopkins University School
of Medicine, Baltimore, MD, USA
SHANNATH L. MERBS • Wilmer Eye Institute, Johns Hopkins University
School of Medicine, Baltimore, MD, USA
SALLY A. MOODY • Department of Anatomy and Regenerative Biology,
The George Washington University School of Medicine and Health Sciences,
Washington, DC, USA
JACOB NELLISSERY • Neurobiology Neurodegeneration and Repair Laboratory,
JOHN M. NICKERSON • Department of Ophthalmology, Emory University,
Atlanta, GA, USA
VERITY F. OLIVER • Wilmer Eye Institute, Johns Hopkins University School
of Medicine, Baltimore, MD, USA
YI PAN • Center for Molecular and Human Genetics, The Research Institute
at Nationwide Children’s Hospital, Columbus, OH, USA
Contributors xiii
GUANG-HUA PENG • Department of Ophthalmology and Visual Sciences,

Washington University School of Medicine, St. Louis, MO, USA
RINKI RATNA PRIYA • Neurobiology Neurodegeneration and Repair Laboratory,
ZHAO QIN • Department of Molecular, Cellular, and Developmental Biology,
University of Michigan College of Literature, Science, and the Arts,
Ann Arbor, MI, USA; Developmental Genetics Program, Skirball Institute
of Biomolecular Medicine, New York University School of Medicine,
New York, NY, USA
HARSHA KARUR RAJASIMHA • Neurobiology Neurodegeneration and Repair Laboratory,
RAJESH RAMACHANDRAN • Department of Biological Chemistry, Molecular &
Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA
PAMELA A. RAYMOND • Department of Molecular, Cellular, and Developmental Biology,
University of Michigan College of Literature, Science, and the Arts,
Ann Arbor, MI, USA
T. MICHAEL REDMOND • National Eye Institute, National Institutes of Health,
Bethesda, MD, USA
THOMAS A. REH • Department of Biological Structure, Institute for Stem
Cells and Regenerative Medicine, University of Washington, Seattle, WA, USA
AARON REIFLER • Department of Biological Chemistry, Molecular & Behavioral
JEROME E. ROGER • Neurobiology Neurodegeneration and Repair Laboratory,
JOSHUA SAMMONS • Department of Cell Biology, University of Alabama
ANAND SWAROOP • Neurobiology Neurodegeneration and Repair Laboratory,
SCOTT TAYLOR • Department of Ophthalmology and Visual Sciences,
SARIKA TIWARI • Department of Biology & Center for Regenerative Biology
and Medicine, Indiana University-Purdue University Indianapolis,
ANNA LA TORRE • Department of Biological Structure, Institute for Stem
Cells and Regenerative Medicine, University of Washington, Seattle, WA, USA
JIN WAN • Department of Biological Chemistry, Molecular & Behavioral
SHU-ZHEN WANG • Department of Ophthalmology, University of Alabama
MENGQING XIANG • Department of Pediatrics, Center for Advanced Biotechnology
and Medicine, Graduate Program in Molecular Genetics, Microbiology
and Immunology, UMDNJ-Robert Wood Johnson Medical School,
Piscataway, NJ, USA
RUN-TAO YAN • Department of Ophthalmology, University of Alabama
xiv Contributors
XIAN-JIE YANG • Department of Ophthalmology, Molecular Biology Institute,

Jules Stein Eye Institute, David Geffen School of Medicine, University of California,
Los Angeles, CA, USA
XIN-CHENG YAO • Department of Biomedical Engineering, University of Alabama
at Birmingham, Birmingham, AL, USA; Department of Vision Sciences,
University of Alabama at Birmingham, Birmingham, AL, USA
DONALD J. ZACK • Wilmer Eye Institute and Departments of Molecular Biology
and Genetics, Neuroscience, and Institute of Genetic Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD, USA
Part I
Manipulation of Gene Expression In Vivo

Chapter 1
Conditional Control of Gene Expression in the Mouse Retina

Qian Ding and Lin Gan
Abstract
Conditional knockout is a powerful research tool for specific deletion of target genes, especially for the
genes that are widely expressed and developmentally regulated. The development of the retina involves
multiple intrinsic and extrinsic factors, many are required for embryonic development or expressed in
multiple tissue or cell types. To study their roles in a spatial- or temporal-specific fashion, Cre/loxP-based
gene-targeting approach has been utilized successfully. This chapter describes the methodology of conditional
knockout approach in studying the development of the retina, using LIM homeobox gene Isl1 as an example.
It provides details on targeting vector design and construction, introducing the vector into embryonic
stem (ES) cell, screening ES cell for the recombination events, injecting ES cells, and generating chimeric
and null mice. It also discusses the current issues in the use of Cre/loxP-based gene-targeting approach.
Key words: Mouse, Gene targeting, Conditional knockout, Retinal development, Cre recombinase,
ES cell
1. Introduction
Gene targeting is a powerful tool in studying in vivo function of

mammalian genes. It allows researchers to generate a variety of
mutations at specific murine genomic loci and investigate the gene
function in a physiological context during development and adult
stages. However, many genes have roles in multiple tissues or
organs and are required for normal embryogenesis and survival.
Their disruption in the germline often causes early embryonic
lethality, preventing analysis at later stages (1). Moreover, deletion
of gene in multiple cells and tissues where its function is required
also renders problems of interpreting the function of the gene in a
system or cell type as the phenotype of germline deletion due to
cell-autonomous mechanisms rather than a combination of effects
Shu-Zhen Wang (ed.), Retinal Development: Methods and Protocols, Methods in Molecular Biology, vol. 884,
DOI 10.1007/978-1-61779-848-1_1, © Springer Science+Business Media, LLC 2012
3
4 Q. Ding and L. Gan
in more than one tissue or cell type (2). To bypass these limitations,
the site-specific recombination system is utilized to restrict the
gene deletion at specific stages as well as in cell type- or tissue-
specific fashion. Cre/loxP recombination is one of the most widely
used and best refined system to generate conditional knockout
mice to date (3).
Cre recombinase (Cre) is a 38-kDa protein from P1 bacterio-
phage that recognizes and catalyzes homologous recombination
between two 34-bp loxP (locus of crossover in P1) sites, resulting
in an excision of a fragment of DNA flanked by loxP sites (Fig. 1) (4).
The Cre recombinase can be expressed under the control of cell- or
tissue-specific promoters. Cre mice are generated by either trans-
genic or gene-targeting (knock-in) strategy. loxP sites are introduced
Fig. 1. Schematic diagram of tissue-specific controlling of gene expression by Cre recom-

binase. (a) The 34-bp loxP site consists of two inverse repeats (arrow) flanking an 8-bp
core sequence (box) which confers the directionality of the site. (b) Cre recombinase
(gray)- mediated gene excision between two loxP sites. (c) Generation of conditional
knockout mouse by breeding a mouse with homozygous floxed gene (left) with a mouse
carrying tissue-specific Cre (right). After two generations, the offspring carrying both
homologous floxed gene and Cre have tissue-specific disruption of the targeted gene in
Cre-expressing tissue. Checked box, homologue arms; open box, targeted gene; black
triangle, loxP sites.
1 Conditional Control of Gene Expression in the Mouse Retina 5
to the genome at desired sites by homologous recombination

with targeting vector in embryonic stem (ES) cells. Then the loxP
carrying mice generated from the ES cells are bred with Cre mice.
The offspring carrying both loxP and Cre alleles will undergo the
excision of DNA fragment from their genome in specific cells or
tissues where Cre is expressed. In recent years, as more and more
regulators in mouse retinal development have been identified,
many Cre mouse lines have been established to exert the deleter
function in the developing or the mature retina.
The mouse retina develops from optic vesicle (OV), a protrusion
of the neuroepithelium of the diencephalon at embryonic day 8.
The distal OV invaginates to form optic cup with the inner layer
developing into neural retina (5). Birthdating analysis revealed that
the retinal progenitor cells pass through a series of competent
stages and generate different retinal cell types in a phylogenetically
conserved order, with retinal ganglion cells, amacrine cells, hori-
zontal cells and cone cells born before birth, and rod, bipolar, and
Müller cells born after birth (6). Choosing a suitable mouse line
with specific expression of Cre in retinal cell types of interest is
necessary to conditionally inactivate a gene and elucidate its role of
in the neural retina. This chapter will use the Isl1 conditional
knockout mouse as an example to illustrate the use of the Cre/loxP
gene-targeting strategy in retinas. We will describe the general
approach to the generation of conditional knockout mice for the
study of gene function in the developing mouse retina and address
current issues in gene targeting.
2. Materials
2.1. ES Cells 1. W4 ES cells.

and Mouse Lines 2. C57BL/6J mice.
3. ROSA26-FLPe mice.
2.2. ES Cell Culture 1. Dulbecco’s minimal essential medium (DMEM) with high
and Growth Condition glucose, 15% fetal bovine serum (FBS) (heat activated at 56°C,
(W4 ES Cells) 30 min), 2 mM glutamine (from 100× stock), 0.1 mM nones-
sential amino acids (from 100× stock), 50 mg/ml penicillin and
streptomycin (aliquoted and stored at −20°C), and 1,000 U/ml
LIF, 1 mM b-mercaptoethanol (aliquoted and stored at −20°C).
2. 0.1% Trypsin in PBS (store at 4°C).
3. 0.1% Gelatin in water (sterilize by autoclaving, store at 4°C).
4. Tissue culture plates with fibroblast feeder cells.
5. 2× Freezing medium: 60% DMEM, 20% FBS, and 20% DMSO.
6. Cell culture facility equipped with laminar flow cabinet,
humidified incubator (5% CO2:95% air, 37°C).
2.3. Electroporation 1. Electroporation apparatus.

2. Electroporation cuvettes.
3. Growing ES cells.
4. ES cell medium.
5. Gelatinized plates.
6. 200 mg/ml G418 in PBS.
7. 0.05% Trypsin in saline/EDTA.
2.4. Southern Blot 1. ES cell lysis buffer: 10 mM Tris–HCL, pH 7.5, 10 mM EDTA,

10 mM NaCl, 0.5% sarcosyl, and 1 mg/ml Proteinase K.
2. NaCl–ethanol solution: 660 ml of 5 M NaCl mixed with 50 ml
100% ethanol.
3. Restriction enzyme (RE) digestion cocktail: RE buffer, 10 mM
spermidine, 0.2 mg/ml BSA, and 40 U RE each.
4. 0.8% Agarose. 1× TAE gel running buffer with ethedium
bromide (EtBr).
5. Denature buffer: 0.1 M HCl.
6. Transfer buffer: 0.4 M NaOH.
7. Washing buffer: 2× SSC, 0.1% SDS in water.
8. Phosphorimaging system.
2.5. Microinjection 1. Blastocysts from pregnant female mice.

2. Microinjection medium: Hepes-buffered DMEM, 5% FBS, ali-
quoted and stored at −20°C.
2.6. Embryo Transfer 1. Embryo transfer hosts (female mice at 2.5 days of
pseudopregnancy).
2. Anesthetics (avertin): dissolve 1 g of tribromoethanol in 80 ml
of distilled water by gentle warming (40°C), add 1 ml of ter-
tiobutyl alcohol, and mix well. Store at 4°C in dark.
3. Sterile surgical instruments.
4. Embryo transfer pipette.
5. Mouth pipette.
6. Anesthesia.
7. 70% Ethanol.
8. Wound clips.
2.7. Genotyping 1. Tail digestion buffer: 10 mM Tris–HCL (pH 8.0), 25 mM

EDTA (pH 8.0), 100 mM NaCl, 1% SDS, and 0.2 mg/ml
proteinase K.
2. Tail restriction digestion cocktail for Southern: 5–10 mg tail
DNA, 3 ml of 10× RE buffer, 0.3 ml of 0.1 M spermidine,
0.3 ml of 2 mg/ml BSA, 40 U RE, and add H2O to 30 ml.
3. PCR mixture: 1 mg DNA as template, 1× PCR buffer,

200 mM each dNTP, 1 U Taq polymerase in a 25 ml volume,
10 pmol each oligonucleotide primer 5¢-GGTGCTTAGCGGT
GATTTCCTC and 5¢-GCACTTTGGGATGGTAATTGGAG
to detect a 452-bp product of wild-type Isl1 allele and a 512-
bp product of Isl1CKO allele.
3. Methods
3.1. Vector Design The detailed information of a targeting vector is not within the
and Construction scope of this chapter. In short, a targeting vector is composed of
sequences homologous to those in the desired genomic integration
site, consisting of upstream homologous region, the upstream loxP
site, the region to be disrupted, the downstream loxP site, and the
downstream homologous region, positive (to select the clones with
vector DNA incorporated) and negative (to kill the clones with the
whole vector integrated) selection markers, and plasmid backbone
(7) (see Note 1). loxP sites are introduced into a targeting vector
so that they flank the genomic region to be deleted. It is crucial
that these insertions will not interfere with the normal expression
of the gene. Therefore, loxP sites are often placed in introns with-
out disrupting the splicing, and the mouse carrying the targeted
gene is phenotypically wild type (see Note 2) (Fig. 1).
Here, we describe the generation of Isl1 conditional knockout
mice. ISL1 is one of the founding members of the LIM-
homeodomain transcription factor family. Conventional Isl1 knock-
out mice do not survive beyond E11.5. To assess the role of ISL1
in the retina development in the mid- to late gestation stages, an
Isl1 conditional knockout (Isl1CKO) allele was generated (8). Among
the four exons, Exon 2, which encodes the first LIM domain, was
selected to be disrupted. To construct the targeting vector, we
inserted a neomycin-resistance gene cassette flanked by FRT sites
along with two loxP sites, one at the 5¢ end of Exon 2 and the other
at the 3¢ end of Exon 2 (Fig. 2).
3.2. Manipulation 1. Gelatinize plates: add 5 ml of gelatin solution to each 10-cm

of Mouse ES Cells plate and leave the plates at room temperature for 20 min.
Aspirate off the gelatin and allow to air-dry.
2. Make feeder plates: start mouse fibroblast cell (STO cell) culture
in a 10-cm plate with 15 ml of medium, add 6–10 ml of mito-
mycin C to the medium, and incubate the plate at 37°C for
2–3 h. Rinse with PBS twice, trypsinize and plate STO cells
onto the gelatinized plates at a density of 4 × 106/10-cm plate.
3. Thaw an ES cell vial and plate on the feeder plate at a density
of 2 × 106/10-cm plate (see Notes 3 and 4).
4. Change medium daily.
Fig. 2. Generation of Isl1 conditional knockout allele. Isl1 genomic structure and restriction
enzyme map is shown at the top. White boxes are exons. Thick bars are the sequences
used to generate homologous arms in the targeting vector; open arrowhead, FRT site for
flipase recognition; solid arrowhead, loxP site for Cre recombinase recognition. Neo PGK-
neomycin-resistance gene, DTA diphtheria toxin gene for negative selection of ES cells.
3.3. Electroporation 1. Linearize 200–300 mg of Isl1 conditional knockout targeting

of ES Cells vector DNA purified by CsCl gradient centrifugation method
by Not I digestion, followed by extraction with phenol/
chloroform, precipitation with two volumes of ethanol, washing
with 70% ethanol. Resuspend the DNA in sterile TE or PBS at
a concentration of 1 mg/ml.
2. Feed ES cells 3 h before harvesting. After collecting ES cells,
wash the cell pellet twice with PBS. Resuspend ES cells in PBS
to make the concentration of about 2 × 107/0.75 ml.
3. Add 20 mg of DNA to each 0.75 ml of cells and electroporate
the mixture at 0.25 kV, 500 mF. After electroporation, place
the mixture on ice for 10 min and plate out the cells on five
plates seeded with mitomycin C-treated STO cells.
4. From the following day, feed the cells daily with ES medium
supplemented with G418 at 200 mg/ml. Colonies should be
visible 6 days after electroporation and picked up at 7–9 days
after electroporation.
3.4. Screening for 1. Prepare 96-well plates with feeder cells.

Targeted ES Cells by 2. Wash the ES cell-containing plates twice with PBS. Leave cells
Southern Blot Analysis in PBS during picking. Pick the ES cell clones with tips under
a dissecting microscope and transfer each colony into one well
of 96-well plate containing 35 ml of 0.05% trypsin at room

temperature. Digest the colonies for 10–20 min in an incuba-
tor, add 80 ml of ES medium to neutralize the trypsin, mix well
by pipetting, and transfer the cells to two or three 96-well
plates with feeder cells. Change medium after 6 h and daily
thereafter.
3. When the colonies in 96-well plates have reached at least 80%
confluence, freeze the clones in one set of plates with freezing
medium and keep the plates in −70°C freezer. Wash the cells
in the other sets twice with PBS and add 50 ml of ES cell lysis
buffer and incubate overnight at 55–60°C in a humid
atmosphere.
4. Cool the plates on ice, then add 150 ml of NaCl–ethanol mix
to each well to precipitate the DNA. Let the plate stand on the
bench for 1 h. Discard the solution by inverting the plate, and
add 150 ml of 70% ethanol to wash the wells. Invert the plates
to discard the ethanol and briefly blot on paper towels. Repeat
the washing step once. Air-dry the plates.
5. Make digestion cocktail and add 30 ml to each well. Incubate
the plate in 37°C overnight in humid atmosphere.
6. Next day, add loading buffer, mix well, run the samples in 0.8%
agarose gel. Treat the gel with 0.1 N HCl for 15–30 min with
shaking, vacuum transfer the DNA with 0.4 M NaOH for 4 h
to overnight to Hybond-N+ membrane. After transferring,
briefly rinse the filter in Tris–HCl, pH 7.4, add Rapid Hyb and
probe, and hybridize at 65°C in Hybaid Oven for overnight.
Wash the filter with 2× SSC–0.1% SDS twice and image by
phosphorimaging system.
3.5. Blastocyst 1. Harvest E3.5 blastocysts from pregnant females.

Injection 2. Feed ES cells in growth phase for 1 h. Trypsinize the ES cells to
single cells and resuspended in microinjection medium at 4°C.
3. Pick up a blastocyst with a micropipette and place it in the
center of the field under the microscope.
4. Pick up 6–8 small round ES cells in the injection pipette. Place
the injection pipette in the same focal plane as the blastocyst.
5. Apply force to pop the injection pipette through the trophec-
toderm wall.
6. Expel ES cells into the blastocoel cavity and remove the pipette.
7. Repeat this procedure until all the embryos are injected with ES
cells and transfer the blastocysts to the culture medium for a
brief period of culture in a 5% CO2:95% air in 37°C incubator.
3.6. Embryo Transfer 1. Mate female mice with sterile males during oestrus to produce
pseudopregnant mice, which serve as embryo transfer hosts on
the third day of pseudopregnancy.
2. Weigh and anesthetize the recipient female with avertin.

3. Take up a column of air, medium, a small air bubble, 10–15
embryos to be transferred, and finally a small air bubble at
the tip.
4. Clean the back of the mice with 70% ethanol and make a 1-cm
transverse incision at the level of the first lumbar vertebra. Slide
the incision to one side and cut the peritoneum wall. The
uterus is exteriorized and the reproductive tract is pulled out
with forceps.
5. Insert the tip of the transfer pipette into the lumen and expel
the embryos with the air bubble as a marker showing the
expulsion of the embryos.
6. Gently return the reproductive tract to the body cavity and
repeat on the other side.
7. Close the skin incisions with a small wound clip.
3.7. Determination 1. A convenient way to determine the contribution of targeted

of Chimerism ES cell in the offspring is to monitor the coat color. For example,
W4 ES cells derived from agouti 129S6 mice are injected into
blastocysts from albino C57BL/6J-Tyrc-2J mice to produce
mouse chimeras with agouti/white coat color. Generally the
degree of coat color chimerism represents the degree of contri-
bution of germline.
2. ES cell lines commonly used are often derived from male
embryos and the resulting chimeras are expected to be males.
3. Choose the highly chimeric animals to breed to mice of appro-
priate genetic background to obtain F1 heterozygotes for the
Isl1CKO allele (see Note 5). Besides producing F1 heterozy-
gotes, this mating also confirms the germline transmission in
the chimera by the genetic markers such as coat color and eye
color of the offspring. The pups from W4 ES cell-derived
gametes will be observed with black eye at birth and the agouti
phenotype at around day 7. Half of the progenies with
germline transmission are expected to be heterozygote and are
determined by Southern blot or PCR genotyping methods.
3.8. Southern Blot 1. Collect £1 cm of mouse tail into a 1.5-ml tube (day 8–10 pups
and PCR Genotyping are ideal).
2. Add 600 ml of digestion solution to the tail and incubate at
55°C with shaking for ³4 h (best over night).
3. Let the tubes cool to room temperature. Extract with 500 ml
phenol/chloroform (1:1) and transfer about 400 ml top layer
with a wide-bore tip to a fresh tube.
4. Add 1 ml of ethanol, mix and precipitate the DNA for 1–2 min
at room temperature, and wash DNA once with 70% ethanol.
5. Dry the DNA and dissolve in 50 ml of TE. The DNA

concentration should be about 1 mg/ml.
6. Southern blot of tail DNA samples was performed as described
in Subheading 3.2.
7. PCR genotyping was carried out using the following condi-
tions: denaturation at 95°C for 5 min, followed by 35 cycles of
95°C for 30 s, 55–70°C for 30 s, and 72°C for 30 s.
3.9. Tissue-Specific 1. Cross the heterozygous mice for the floxed gene to ROSA26-
Deletion FLPe mice to remove FRT-flanked neomycin-resistance gene.
2. Breed the mice without neo to Six3-Cre mouse line. Six3-Cre
mice express Cre recombinase in the eye field and the ventral
forebrain starting at E9–E9.5 and have been used successfully
as an effective retina-specific deleter (9). In our experiments,
Cre recombinase-mediated deletion of the loxP-flanked Isl1
Exon 2 resulted in a null mutation via a reading frame shift (see
Notes 6–10).
3. After one more generation, the progenies carrying homozy-
gous Isl1loxP/loxP and Six3-Cre have Isl1 deleted in a tissue-specific
fashion (Fig. 3a). Usually, several breeding schemes can be
developed, depending on the viability and fertility of each gen-
otype. We incorporated a lacZ knock-in allele, an Isl1 null
mutant, into the breeding scheme. Thus, only one floxed gene
needs to be excised by Cre recombinase to produce Isl1-null
cells. Moreover, the knock-in lacZ reporter gene is used to
trace Isl1-expressing cells (Fig. 3b) (see Note 11).
4. Notes
1. The construction of targeting vector is very important for the

targeting efficiency (10). Usually longer homology arms
increase targeting frequency (11, 12). The vector should be
constructed from a DNA library that originates from the
isogenic mouse strain that the ES cell line is derived. Base-pair
mismatches could strongly affect the efficiency of intrachro-
mosomal recombination in mammalian cells (13, 14). Besides,
the targeting efficiency also has locus-specific variations
depending on additional parameters, such as transcriptional
activity, chromosomal location of the target genes, and spatial
aspects of the nucleus (10).
2. When the loxP sites are inserted into the genomic loci, the
selection of the region to be floxed is very important for both
deletion and recombination efficiency. Usually the further the
two loxP sites are apart, the less frequently the recombination
Fig. 3. Breeding scheme of Isl1 conditional knockout. (a) Heterozygous breeding scheme.
After two generations, F3 mice carrying homozygous for the floxed Isl1 and Six3-Cre locus
are present as Isl1 conditional knockout. (b) Incorporation of lacZ allele into the breeding
scheme. F3 mice harboring a null allele and a floxed allele undergoing excisional recom-
bination by Six3-Cre recombinase.
event happens (15). Meanwhile the floxed sequence is also

needed to be vital for the expression of the target gene. So
theoretically, the segment with minimal length, which can lead
to the disruption of the gene expression, should be selected
(10). After the conditional knockout mice are generated, the
deletion efficiency should be examined by in situ hybridization
at mRNA level or immunohistochemistry at protein level.
3. The protocols listed in Subheading 3 provide only the guide-
lines for ES cell culture and necessary adjustments should be
made daily according to ES cell growth. ES cell medium
should be as fresh as possible and only medium less than
2 weeks old is used. Old leftover medium can be saved for
STO cell culture.
4. ES cells should be split every 3 days or less. Growing ES cells
undisturbed for 3 days could result in the differentiation.
5. ES cells from the 129 mouse strains were most widely used;
however, different genetic background may affect how close
the model is to a desired purpose. In addition, a uniform genetic

background allows the precise comparison of the mutant phe-
notypes to the control. Backcrossing is the straightforward way
and is applicable to any inbred strain (16, 17). Usually the
heterozygotes confirmed by Southern blot are crossed with
mice of an inbred strain, and the heterozygous progenies from
this mating are then backcrossed to the mice of the inbred stain
again. Usually after seven generations of backcrossing, 99% of
loci not linked to the mutant allele will be homozygous (18).
6. Cre mice can be generated by both transgenesis or through a
knock-in strategy. Transgenic mouse is easier and faster to
obtain, but due to the poorly defined promoter elements used
to drive Cre expression, the specificity of the transgene expres-
sion is not very reliable (19). The use of knock-in approach by
homologous recombination can ensure more faithful and
regulated expression, but if the expression level from the promoter
is low, the deletion will not be efficient enough. In the case of
Isl1 conditional knockout retina, Six3-Cre can delete Isl1 in
more than 90% of the cells from onset of Isl1 expression, while
we did not see the deletion until late embryonic stage by
Math5-Cre mice ((8); unpublished observations by Ling Pan,
University of Rochester).
7. Before the application of a newly generated Cre line, the spatial
and temporal pattern of the recombination should be eluci-
dated first. Breeding the Cre mouse with a reporter line such as
the Z/EG mice (20). By observing the activation of reporter,
we can monitor the Cre-mediated recombination events.
8. Besides crossing with Cre mice in retina-specific knockout,
Cre recombinase can also be delivered by injecting the Cre-
expressing virus directly into the vitreous cavity (21).
9. The targeted gene in conditional knockout mice can also be
inactivated by inducible Cre. This induction is mediated by a
ligand-binding reaction (22). Cre is expressed as a fusion pro-
tein with a mutated ligand-binding domain of the estrogen
receptor and can be specifically activated by inducer, tamox-
ifen, a synthetic estrogen analog. Thus the gene inactivation
depends on the temporal course of inducer administration.
10. For each conditional mouse line, several Cre lines can be
chosen (Table 1). Even targeting the same cell type, different
Cre lines may have different performance and onset time. In
addition, using Cre lines for specific cell type can help to iden-
tify the cell-autonomous role of the targeted gene.
11. According to our breeding strategy showing in figure 3, 12.5%
of the offspring will be conditional knockout mice. This per-
centage is under the assumption that the targeted gene and the
Cre loci are not linked.
Table 1
Mouse lines expressing Cre recombinase in the developing
retina
Promoter Targeted cell types References
Pax6 Distal neural retina (23)

Six3 Neural retina (9)
Chx10 Neural retina (24)
Math5 RGC, amacrine, and horizontal cell (25)
Thy1.2 RGC and neural retina (26)
mRho Rod bipolar cells and rod photoreceptor (27)
Nes Muller glia (28)
Foxg1 Muller glia (29)
Pcp2 Bipolar cells (30)
ChAT Starburst amacrine cell (31)
Ptf1a Amacrine and horizontal cell (32)
Pax6 paired box gene 6, Six3 six/sine oculis subclass of homeobox gene,
Math5 murine atonal homolog 5, Thy1.2 thymus cell antigen 1.2, mRho
mouse rhodopsin, Foxg1 Forkhead box G1, Pcp2 purkinje cell protein 2,
ChAT choline acetyl transferase, Ptf1a pancrease-specific transcription factor
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Chapter 2
Generation of Transgenic Xenopus Using Restriction

Enzyme-Mediated Integration
Mohammad Haeri and Barry E. Knox
Abstract
Transgenesis, the process of incorporating an exogenous gene (transgene) into an organism’s genome, is
a widely used tool to develop models of human diseases and to study the function and/or regulation of
genes. Generating transgenic Xenopus is rapid and involves simple in vitro manipulations, taking advantage
of the large size of the amphibian egg and external embryonic development. Restriction enzyme-mediated
integration (REMI) has a number of advantages for transgenesis compared to other methods used to pro-
duce transgenic Xenopus, including relative efficiency, higher transgene expression levels, fewer genetic
chimera in founder transgenic animals, and near-complete germ-line transgene transmission. This chapter
explains the REMI method for generating transgenic Xenopus laevis tadpoles, including improvements
developed to enable studies in the mature retina.
Key words: Transgenesis, Transgene, Reporter Gene, Cell-specific promoter, Gene expression,
Xenopus laevis, Amphibians, Gene regulation, REMI
1. Introduction
Xenopus is a primary animal model in use for decades to under-

stand vertebrate development (1), nuclear reprogramming (2),
and metamorphosis (3, 4). In addition, Xenopus has been an
extremely important system for elucidating the cell (5, 6), molecu-
lar (1) and circadian (7–10) biology of the retina, and is emerging
in utilization for investigating retinal diseases (11–18) and regen-
erative mechanisms (19). The study of promoters and gene regula-
tion are other applications of transgenesis technology (20–23).
During the last two decades, a number of different transgenesis
methods have been described; they include those mediated by
restriction enzymes (24, 25), I-SceI meganuclease (26–28), trans-
posons (29–31), phi-C31-integrase (32–34), and DNA injection (1).
Shu-Zhen Wang (ed.), Retinal Development: Methods and Protocols, Methods in Molecular Biology, vol. 884,
DOI 10.1007/978-1-61779-848-1_2, © Springer Science+Business Media, LLC 2012
17
18 M. Haeri and B.E. Knox
The advantages and drawbacks of the various transgenic Xenopus

methods have recently been reviewed (35). Typically, restriction
enzyme-mediated integration (REMI) is able to produce transgenic
embryos easily without a large investment in expensive equipment
or time-consuming training in specialized techniques. When a
fluorescent reporter transgene is used, then embryos can be sorted
early, e.g. neurulation, if a suitable promoter is used. Typically,
between 20 and 50 transgenic tadpoles with uniform (not genetic
chimera) expression patterns can be generated in a single day.
However, the integration site of the transgene is random and copy
number is variable between different primary transgenic animals
(36). Natural mating of F0 animals can be performed to create
transgenic lines, often with less mosaicism and a high-level expres-
sion pattern than the original animals.
Overview. REMI transgenesis in Xenopus requires five steps:
1. Preparation of interphase egg extract (37). Eggs from hormon-
ally stimulated females are collected and a cytoplasmic fraction
is prepared after they have progressed in vitro into interphase.
The extract is used to initiate decondensation of sperm chro-
matin and swelling of sperm nuclei, which is visible under a
microscope. The egg extract is prepared in advance and stored
at −80°C for at least 6 months.
2. Preparation of sperm nuclei (37). Intact sperm are isolated from
whole minced testes of fully mature male frogs. Nuclei are pre-
pared by hypotonic treatment of sperm and permeabilized with
a mild detergent (lyso-PC) allowing egg extract, plasmid DNA,
and restriction enzymes access to sperm chromatin in the
REMI reaction. The sperm nuclei can be prepared in advance
and stored at −80°C for at least 1 year.
3. Restriction enzyme-mediated integration reaction (25, 38).
Permeabilized sperm nuclei are mixed with egg extract, linear-
ized plasmid containing the transgene cassette, and restriction
enzyme. The partially decondensed chromatin is rendered
accessible to restriction enzyme generating breaks that can
anneal with linearized plasmid. DNA ligase and repair activities
in the egg cytoplasm (39) link the plasmid DNA into chroma-
tin and closes restriction enzyme breaks. The reaction is carried
out for several minutes before nuclear transplantation.
4. Microinjection of sperm nuclei (2). After the REMI reaction,
sperm nuclei are transplanted into eggs. The glass needle must
be chosen large enough to minimize damage to the fragile
decondensed sperm nucleus while small enough to minimize
trauma to the egg. A successful transplantation will generate a
transgenic embryo expressing the gene of interest in a significant
fraction of the injected eggs. The majority of integrations occur
during the REMI and before the fertilization of the egg, thus
2 Generation of Transgenic Xenopus Using Restriction¼ 19
generating non-chimeric embryos. Although it is rare, we have

observed transgenic tadpoles expressing the transgene
unilaterally.
5. Selection of transgenic embryos. The final step is to select fertil-
ized eggs and to sort out well-developing embryos at gastrula-
tion, neurulation, etc. Cleaving transplanted embryos should
be carefully transferred into fresh media with large-bore col-
lecting pipettes. Often, a small tissue extrusion (bleb) on an
otherwise normally developing embryo is observed on the sec-
ond day after injection (dpi). These blebs are outpouching cells
from the puncture hole in the vitelline membrane created dur-
ing the injection. Although these blebs can be manually
removed, it is preferable to allow them to fall off spontane-
ously, which usually occurs after neurulation. Eggs that receive
damaged sperm nucleus, no nucleus, or more than one nucleus
will exhibit aberrant cleavage patterns (multiple cleavage
planes, partial or unilateral cleavage, pseudocleavage, and
incomplete or shallow furrow). It is imperative to continuously
sort normally developing embryos from the maldeveloped
ones because the latter are easily infected and will compromise
the healthy embryos. Typically, up to 50% of embryos are lost
at each major developmental stage (i.e. gastrulation, neurula-
tion, and feeding larvae). Approximately 30% of the tadpoles
are transgene positive by genotyping a week after nuclear trans-
plantation. Selection of tadpoles by transgene fluorescence will
depend upon the promoter, level of expression, mosaicism,
and stability of the particular gene chosen for study.
The number of integration sites as well as the number of trans-
genes per site (concatemers) may vary between one and more than
ten, with some influence exerted by the amount of DNA and the
restriction enzyme used in the REMI reaction. The exact transgene
number and sites of integration can be determined by Southern
blotting (36) while the transgene expression level can be accurately
measured by any number of quantitative methods such as real-time
PCR. Since transgenes are typically integrated as concatemers, the
addition of more than one type of linearized plasmid with compat-
ible ends will produce transgenic tadpoles expressing multiple dif-
ferent plasmids with a high frequency (over 90%).
Critical elements for high-yield transgenesis are: (1) high-
quality eggs (e.g. have a clear maturation spot, even animal pole
coloration and easily distinguishable equatorial border); (2) high-
quality sperm nuclei (e.g. well permeabilized and concentrated at
5–8 × 107 nuclei/ml); (3) high-quality egg extract (e.g. creates an
easily observable swelling of the sperm within a few minutes at
room temperature); (4) highly purified linearized DNA (preferably
prepared immediately before use).
2. Materials
2.1. Trangenesis 1. Pregnant mare serum gonadotropin (PMSG): store PMSG

aliquots of 100 U/100 μl dH2O at −20°C. On the day of
2.1.1. Hormones
injection, dilute one aliquot of PMSG in 400 μl sterile MilliQ
dH2O for each frog (see Note 1).
2. Human chorionic gonadotropin (HCG): store HCG as pow-
der in 500–700-U aliquots at 4°C. Dissolve 500–700 U of
HCG in 500 μl of diluent provided by the manufacturer on the
day of injection. Do not store dissolved HCG (see Note 2).
2.1.2. Stock Solutions 1. 10× MMR: 1 M NaCl, 20 mM KCl, 20 mM CaCl2(2H2O),

10 mM MgCl2(6H2O), and 50 mM HEPES(–Na). Adjust pH
to 7.8 with 10 N NaOH. Bring volume up to 3 L with MilliQ
dH2O. Dispense into 1-L bottles, autoclave, and store at 4°C.
2. 8× Egg laying solution: 0.88 M NaCl, 16 mM KCl, 4.8 mM
Na2HPO4, 0.25 M Tris base, 16 mM NaHCO3, 16 mM MgSO4
anhydrous, and 25 ml acetic acid. Adjust pH to 7.6 if necessary
with 10 N NaOH or acetic acid. Bring volume up to 4 L with
MilliQ dH2O. Pour into a container and store at 4°C.
3. 2% Cysteine solution in 1× MMR: dissolve L-cysteine hydro-
chloride monohydrate (Sigma C7880–500G) in 1× MMR
(2%). Adjust pH to 7.9 with sodium hydroxide pellets. Bring
volume up to 400 ml with 1× MMR.
4. 100 mM MgCl2: dissolve MgCl2 in MilliQ dH2O. Bring vol-
ume up to 100 ml with MilliQ dH2O. Filter-sterilize and store
at room temperature.
5. 6% Ficoll, 0.4× MMR solution: dissolve Ficoll in MilliQ dH2O,
add 10× MMR to reach a final concentration of 0.4× MMR.
Bring volume up to 2 L with MilliQ dH2O. Filter-sterilize and
store at −20°C.
6. 6% Ficoll, 0.1× MMR solution: dissolve Ficoll in MilliQ dH2O,
add 10× MMR to reach a final concentration of 0.1× MMR.
Bring volume up to 2 L with MilliQ dH2O. Filter-sterilize and
store at −20°C.
7. 0.1× MMR/10 μg/ml gentamicin.
8. 0.1× MMR, prepared with sterile MilliQ dH2O.
9. Interphase egg extract aliquot (made and stored at –80°C).
10. Sperm dilution buffer: 250 mM sucrose, 75 mM KCl, 0.5 mM
spermidine, and 0.2 mM spermine. Dissolve sucrose in MilliQ
dH2O. Add in other reagents. Bring volume up to 45 ml. Adjust
pH to 7.3–7.5. Bring volume up to 50 ml with MilliQ dH2O.
Filter-sterilize, dispense into 1-ml aliquots and store at −20°C.
11. Linearized plasmid DNA (linearized, purified, adjusted to a
concentration of 200 ng/μl, and stored at −20°C). Any restriction
enzyme that does not cut frequently might be suitable for

linearization of the plasmid. Our first choice is XhoI, followed
by NheI, NotI, SalI, AgeI, BamHI, EcoRI, and ApaLI.
12. Restriction enzyme, 0.5 U/μl, diluted in compatible buffer.
We usually use XhoI (New England Biolab) and dilute the
enzyme in its 10× compatible buffer (NEB2). The restriction
enzyme should be the same used for the linearization of the
plasmid.
2.1.3. Other Materials 1. Plastic mesh-coated plates: Cut pieces of nylon mesh 600 μm
(http://www.smallparts.com) and attach to bottom of 90-mm
plate using chloroform (perform under hood). Glue the edge
of the plastic mesh with melting plastic glue. Plates can be
reused for many rounds of injections (Fig. 1).
2. Transplantation needles: flame-polish the ends of capillary
pipettes to prevent clogging of the needle by small pieces of
glass during the injection. If the glass pipettes are not silanized,
prepare a silanization chamber; place a pack of glass capillary
tubes (1.2 mm OD and 0.69 ID, Warner Instruments) in a
Fig. 1. Injection of eggs with sperm nuclei. (a) Injection needles with beveled tip. Glass needles are tapered and broken to
create a 40–60-μm diameter beveled opening. (b) Eggs are dispersed in nylon mesh-covered plates along parallel lines
with 4–6 eggs width on the mesh. A total of ~1,500 eggs are placed in each plate. (c) Healthy eggs should have clear matu-
ration spots (arrows) and non-mottled appearance. The image on the right is at a higher magnification. (d) Eggs are injected
around the maturation spot on the animal pole with continuous flow from the needle. The tip of the needle should approach
the injection site and poke the membrane with a quick jabbing motion, penetrating only beyond the membrane as shown.
small beaker 1/3-filled with silane (N,N-dimethyl-

trimethylsilylamine, Sigma) in a dessicator overnight and bake
them at 180°C for 2 h. Using a capillary glass puller, pull
capillary glasses to make needles and clip the tip to create a
40–60-μm diameter opening. The clipping of the tip should be
performed carefully with forceps under the dissecting micro-
scope with a measuring grid to generate beveled tips. The sharp
tip will make the injection of the eggs fast and easy and cause
less damage to the membrane (Fig. 1a).
3. Transplantation unit: prepare a micromanipulator with a glass
pipette holder, an oil-filled infusion pump (Harvard Apparatus),
a glass syringe, and plastic tubing. Assemble the transplantation
unit, fill the syringe with mineral oil and remove any bubbles in
the syringe or along the plastic tubing (2.4 mm OD and 0.78
ID, Fisher). Set the infusion rate of the pump to 40 μl/h.
2.2. Interphase Egg 1. PMSG (see above).

Extract 2. HCG (see above).
2.2.1. Preparations
2.2.2. Stock Solutions 1 M HEPES: dissolve HEPES in MilliQ dH2O. Adjust pH to 8.2
with 10 N KOH. Bring volume up to 500 ml with MilliQ dH2O
and mix thoroughly until the solution is clear. If pH of diluted
(10 mM) HEPES is not 7.7 (due to the pH of dH2O), adjust to
7.7. Filter-sterilize, dispense into 50-ml aliquots, and store at
−20°C (see Note 3).
1. 1.5 M Sucrose: dissolve sucrose in MilliQ dH2O. Bring volume
up to 100 ml with MilliQ dH2O. Filter-sterilize, dispense into
10-ml aliquots, and store at −20°C.
2. 0.5 M EGTA: dissolve EGTA in dH2O. Adjust pH to 7.7 with
10 N KOH. Adjust volume to 100 ml with MilliQ dH2O. Filter-
sterilize, dispense into 10 ml aliquots, and store at −20°C.
3. 1 M CaCl2: dissolve CaCl2 in MilliQ dH2O. Bring volume up
to 100 ml with MilliQ dH2O. Filter-sterilize and store at room
temperature.
4. 1 M MgCl2: dissolve MgCl2 in MilliQ dH2O. Bring volume up
to 100 ml with MilliQ dH2O. Filter-sterilize and store at room
temperature.
5. Protease inhibitors: chymostatin, leupeptin, hemisulfate, and
pepestatin.
(a) Prepare a protease inhibitor solution for each protease
inhibitor; dissolve 50 mg of the protease inhibitor in 5 ml
of DMSO, for a concentration of 10 mg/ml.
(b) Dispense into 50-μl aliquots. Store at −20°C. We purchase
the following protease inhibitors: Chymostatin (Sigma,
C7268), leupeptin, hemisulfate (EMD, 108975), and
Pepestatin A (MPbio, #19536825) (see Note 4).
6. 20× Extract buffer (XB) salt stock: 2 M KCl, 20 mM MgCl2,

and 2 mM CaCl2. Prepare 1 L with MilliQ dH2O. Filter-
sterilize, dispense into 500-ml aliquots, and store at −20°C.
7. Energy mix: 150 mM creatine phosphate, 20 mM ATP, and
20 mM MgCl2. Prepare 10 ml with MilliQ dH2O. Filter-
sterilize (syringe filter), dispense into 500-μl aliquots, and store
at −20°C.
2.2.3. Buffers 1. XB pH 7.7: 1× (1:20 dilutions of 20×) XB salts, 50 mM

sucrose, and 50 mM HEPES (pH 8.2). Prepare 1 L with
MilliQ dH2O.
2. 2% Cysteine solution in 1× MMR: dissolve L-cysteine hydro-
chloride monohydrate (Sigma C7880-500G) in 1× MMR
(2%). Adjust pH to 7.9 with sodium hydroxide pellets. Bring
volume up to 400 ml with 1× MMR (see Note 5).
3. CSF-XB: 1× (1:20 dilutions of 20×) XB salts, 1 mM MgCl2,
10 mM HEPES pH 8.2, 50 mM sucrose, and 5 mM EGTA.
Prepare 500 ml with MilliQ dH2O (see Note 6).
4. 10× MMR: 1 M NaCl, 20 mM KCl, 20 mM CaCl2(2H2O),
10 mM MgCl2(6H2O), and 50 mM HEPES(–Na). Adjust pH
to 7.8 with 10 N NaOH. Prepare 3 L with MilliQ dH2O.
Dispense into 1-L bottles, autoclave, and store at 4°C.
5. 8× Egg laying solution: 0.88 M NaCl, 16 mM KCl, 4.8 mM
Na2HPO4, 0.25 M Tris base, 16 mM NaHCO3, 16 mM MgSO4
anhydrous, and 2.5% (v/v) acetic acid. Adjust pH to 7.6 if nec-
essary with 10 N NaOH or acetic acid. Prepare 4 L with MilliQ
dH2O. Pour into a container (carboy) and store at 4°C.
6. Versilube F-50.
7. Ten adult female frogs (X. laevis).
8. Other materials: 50-ml conical tubes, 14-ml round-bottom
tubes (LPS, L285991), long cotton swabs, JS-13.1 swinging
bucket rotor, 1- and 2-ml syringes, 18-gauge needles, SW 55Ti
swinging bucket rotor, thin wall polyallomer 5 ml Beckman
ultracentrifuge tubes (Beckman #326819), 0.6 ml sterile, low
adhesion microfuge tubes (silanized, LPS), cut 100 μl tips, liq-
uid nitrogen (crushed dry ice as alternative).
2.3. Sperm Nuclei 1. HCG (see above).

2.3.1. Buffer and Reagent
Preparation
2.3.2. Stock Solutions 1. 1 M HEPES: dissolve HEPES in MilliQ dH2O. Adjust pH to

8.2 with 10 N KOH. Bring volume up to 500 ml with MilliQ
dH2O and mix thoroughly until the solution is clear. Filter-
sterilize, dispense into 50-ml aliquots, and store at −20°C (see
Note 7).
2. 0.5 M EDTA pH 8.0: adjust pH with 10 N NaOH. Bring

volume up to 100 ml with MilliQ dH2O. Filter-sterilize and
store at room temp.
3. 1.5 M KCl: dissolve KCl in MilliQ dH2O. Bring volume up to
100 ml with MilliQ dH2O. Filter-sterilize and store at room
temperature.
4. 100 mM DTT (Sigma D0632): dissolve completely in MilliQ
dH2O. Bring volume up to 50 ml with MilliQ dH2O. Filter-
sterilize and store at −20°C.
5. 1% Tricaine (Sigma A5040): dissolve tricaine (ethyl 3-amin-
obenzoate methanesulfonate salt) in MilliQ dH2O. Bring vol-
ume up to 500 ml with MilliQ dH2O. Dispense into 500-ml
bottles and store at −20°C.
6. 10 mM Spermidine trichloride (Sigma S2501): dissolve sper-
midine trichloride completely in MilliQ dH2O. Bring volume
up to 50 ml with MilliQ dH2O. Filter-sterilize and store at
−20°C.
7. 10 mM Spermine tetrahydrochloride (Sigma S1141): dissolve
spermine completely in MilliQ dH2O. Bring volume up to
50 ml with MilliQ dH2O. Filter-sterilize and store at −20°C.
8. 10 mg/ml Leupeptin, hemisulfate (EMD, 108975): dissolve
leupeptin in DMSO. Bring volume up to 5 ml with DMSO.
Dispense into 50-μl aliquots and store at −20°C.
9. 0.3 M PMSF (Sigma P7626): dissolve PMSF completely in
ethanol. Bring volume up to 5 ml with ethanol. Dispense into
100-μl aliquots and store at −20°C (see Note 8).
10. Lysolecithin (Sigma TypeI-L4129): dissolve lysolecithin com-
pletely in MilliQ dH2O. Bring volume up to 5 ml with MilliQ
dH2O. Dispense into 100-μl aliquots and store at −20°C.
11. 10% Bovine serum albumin, FractionV (Sigma A7906): dis-
solve BSA in MilliQ dH2O. Bring volume up to 50 ml with
MilliQ dH2O and store at −20°C.
2.3.3. Buffers 1. 2× Nuclear preparation buffer (NPB): 500 mM sucrose,

30 mM HEPES pH 8.2, 2 mM EDTA pH 8.0, 1.0 mM
Spermidine trichloride, 0.4 mM Spermine tetrachloride, and
2 mM DTT. First dissolve sucrose in ~1,500 ml MilliQ dH2O.
Add the remaining stock solutions. Bring volume up to 2 L
with MilliQ dH2O. Filter-sterilize, dispense into 1 L bottles,
and store at −20°C.
2. 1× NPB, 3% (w/v) BSA, protease inhibitor buffer: 1× NPB,
3% BSA, 10 μg/ml leupeptin, 0.3 mM PMSF. Prepare 50 ml
with MilliQ dH2O in a 50-ml conical tube and keep on ice.
3. 1× NPB, 0.3% (w/v) BSA: 1× NPB, 0.3% BSA. Bring volume
up to 20 ml with MilliQ dH2O. Prepare in a 50-ml conical
tube and keep on ice.
4. Storage buffer: 1× NPB, 0.3% (w/v) BSA, 30% glycerol. Bring

volume up to 10 ml with MilliQ dH2O. Prepare in a 50-ml
conical tube and keep on ice.
5. Sperm dilution buffer: 250 mM sucrose, 75 mM KCl, 0.5 mM
spermidine, and 0.2 mM spermine. Dissolve sucrose in MilliQ
dH2O. Add in other reagents. Bring volume up to 45 ml.
Adjust pH to 7.3–7.5. Bring volume up to 50 ml with MilliQ
dH2O. Filter-sterilize, dispense into 1-ml aliquots, and store at
−20°C.
6. Five to ten sexually mature male frogs (X. laevis).
7. Other materials: JS-13 rotor, 1× MMR, 50-ml conical tubes,
14-ml round-bottom tubes (LPS, L285991), Petri plates, cut
1,000-μl tips, cut 100-μl tips, sterile cheesecloth, small glass
funnel, fine #5 forceps, dissecting tools, pulverized dry ice (on
a tray or in a bucket), 500 μl sterile low adhesion (silanized)
microfuge tubes, pipettors, timer, and two ice buckets.
Notes and Cautions
● Tricaine is a carcinogen. Wear gloves throughout the
procedure.
● All buffers and reagents must remain on ice throughout
the procedure.
● Always use cut tips. Sperm nuclei are sheared if they pass
through an uncut tip.
3. Methods
3.1. Trangenesis 1. Prime 4–6 female frogs (Xenopus laevis) by injecting 100 U of
PMSG into the dorsal lymph sac (Fig. 2a) 4–5 days prior to the
3.1.1. Procedure
HCG injection. Incubate the injected frogs in frog water at
16–18°C (see Note 9).
2. The evening before the day of the transgenesis procedure,
inject each female frog with 500–700 U of HCG and place two
frogs per tank containing frog water. Place the frogs in the
16–18°C incubator overnight. Change the water in the tank
with fresh frog water after the injection. Use a temperature and
(12 h/12 h) light/dark-controlled incubator and set the light
onset at 7:00 am (see Note 10).
3. On the day of the procedure remove frogs from the incubator
and place each frog in a tank filled with 1× egg laying solution
(at frog-room-temperature, 20°C) (see Note 11).
4. Gently collect eggs using a collecting pipette and transfer eggs
from each tank into one 50-ml conical tube. There should be
a maximum of 15 ml of eggs in each tube. If there are more
Fig. 2. Egg extract and sperm nuclei preparation. (a–c) Injection of females with hormones into the posterior lymph sac. Frogs
are covered (including the eyes) with wet paper towel and injected under the skin into the dorsal lymph sac. (d) Washed eggs
are settle to the bottom of the tube. (e) Eggs are treated with cysteine to remove jelly coat. After treatment, eggs are compact
at the bottom of the tube. (f–i) Various stages in the egg extract preparation (see Subheading 3 for details). (f) After the first
spin, eggs are packed but not broken and versilube replacing CSF-XB between the eggs. (g) Crushed eggs separated into
eggs collected from one tank, divide them into two or more
tubes (see Note 12).
5. Decant as much liquid as possible. Wash eggs in 1× MMR
three or more times, until the solution is clear.
6. Remove as much liquid as possible using a collecting pipette
(see Note 13).
7. Fill up the conical tubes with 2% cysteine/1× MMR solution
(to 45 ml line) (Fig. 2b). Put the cap on and gently invert
tubes over and over to strip off the jelly coat from the eggs.
Observe the eggs as they fall to the bottom of the tube. As
their jelly coats are released, the eggs become more compact at
the bottom of the conical tube. For instance, if the initial vol-
ume of eggs was 15 ml, the volume will drop to ~7 ml after
proper dejellying (Fig. 2b). This process should take approxi-
mately 3–5 min depending on the thickness of the jelly coat
and the freshness of the 2% cysteine/1× MMR solution. As
soon as the eggs become compact and their jelly coats are
released, immediately pour off the cysteine and wash eggs
extensively with 1× MMR buffer to remove the cysteine (wash
with 35 ml of 1× MMR buffer five times or more, until the
solution is clear). Frequently, dead eggs (appearing white) will
be found on top of the healthy eggs. Remove these dead eggs
from the top using a collecting pipette (see Note 14).
8. Pour off the 1× MMR and remove the residual MMR by tilting
the tube and removing the solution with a collecting pipette.
9. Add sufficient amount of room-temperature 6% Ficoll/0.4×
MMR (~5 ml) to cover the treated eggs. The hyperosmolar 6%
Ficoll/0.4× MMR solution protects treated eggs, which have
lost their protective jelly coat and will be subjected to damage
to their membrane during the injection.
10. Add 10 ml room-temperature 6% Ficoll/0.4× MMR to mesh-
covered plates and transfer the eggs onto plates (Fig. 2.1b, c):
first swirl the conical tubes to make the eggs float in the solu-
tion, then collect eggs in a plastic pipette, and dispense them
Fig. 2. (continued) three layers: a thick yellow lipid layer on top (L); the desired cytoplasmic layer which appears gray (C); the
bottom layer appears gray/black and consists of unbroken eggs and membranes (M). (h) After the second cytoplasmic spin,
the lipid and cytoplasmic layers are observed and the residual membranes pellet to the bottom. (i) After ultracentrifugation,
the cytoplasm separates into 4 layers. Top layer: the yellow lipid layer (L); second layer: the clear golden cytosol (C); third
layer: membranes and mitochondria (M); bottom layer: glycogen and ribosomes (R). Preparation of sperm nuclei. (j) Testes
are removed and rinsed to remove blood and lipid. (k) Peripheral blood vessels and fat are manually stripped away. (l) Testes
are macerated with fine forceps to release sperm. Sperm are treated with lysolecithin to release nuclei, which are collected
by centrifugation. (m) The sperm nuclear pellet (S) often has a ring of red blood cells (B) at the bottom of the tube, which
should be avoided. (n–q), In order to calibrate the egg extract for nuclear decondensation activity, sperm nuclei (stained with
DAPI) are incubated for various times and amounts of egg extract. Swelling is easily observed in the microscope. Extensive
swelling, such as that shown here in the 5 and 15 min samples should be avoided, and the extract diluted or time adjusted
in the REMI reaction as necessary.
along parallel lines with 4–6 eggs width on the plastic mesh.
Let the eggs settle down and stick to the mesh (5–10 min)
before moving them to the dissecting microscope (this is the
optimal time to perform the REMI reaction).
3.1.2. REMI Reaction 1. In a 1.5-ml silanized tube, first add 5 μl of linearized plasmid
and subsequently add 4 μl of stock sperm nuclei using a cut
pipette tip. Mix gently.
2. Incubate the mixed linearized plasmid and sperm nuclei 5 min
at room temperature.
3. Add the following to the mixed linearized plasmid and sperm
nuclei:
(a) 5 μl Egg extract.
(b) 1 μl of a 1:40 dilution of the restriction enzyme (0.5 U/μl)
in its 10× buffer (see Note 15).
(c) 2 μl of MgCl2 (100 mM).
(d) Bring the volume up to 32 μl with sperm dilution buffer
(15 μl).
4. Warm up the tube containing the reaction between two fingers
and incubate for 3 min.
5. Dilute the reaction with the approximate amount of sperm
dilution buffer (already acclimatized to room temperature)
needed to deliver one sperm nuclei per second based on the
rate of fluid injection in the injector (we usually start with a
sperm nuclei count of 5 × 105/ml, set the flow rate of the injec-
tor (Harvard Apparatus) to 40 μl/h and do a 1:50 dilution of
the REMI reaction with the sperm dilution buffer).
3.1.3. Injection 1. Mix the REMI solution by flicking the tube. Using a pipette
tip fitted with a piece of tygon tubing, draw in 40 μl of the
REMI reaction from the middle portion of the 1.5-ml
microfuge tube (the bottom or the surface of the tube might
contain some unwanted artifacts that clog the needle). Avoid
drawing in air bubbles.
2. Attach a clean and cut needle to the pipette tip and push the
fluid through gently and continuously while keeping the tip of
the needle upright. Looking at the rising level of the solution
in the glass pipette, keep the pressure constant until small fluid
drops come out of the tip of the needle. Remove the needle
from the tube while the pipettor button is still pushed all the
way to prevent adding air bubbles to the needle or drawing the
fluid back into the pipette.
3. Attach the filled needle to the rubber tube connected to the
automated pump (Harvard Apparatus), which is already running.
Check the tip of the needle under the dissecting microscope
before starting injections. The tip of the needle and the fluid
path should be free of blocking artifacts from the REMI or
from pieces of glass. The fluid coming out of the tip should be
visible due to its refraction index difference from the 6%
Ficoll/0.4× MMR. If debris is seen within the glass needle, it
should be changed even if the fluid is moving out of the tip.
Artifacts tend to stick to sperm and this unpredictably reduces
the number of sperm delivered to eggs. When the path is clear
and the fluid is running, place the needle on the manipulator
and tighten it without breaking the glass needle.
4. Inject eggs around the maturation spot on their animal pole
while the fluid is running (Fig. 1e). Approach the tip of the
needle to the injection site and poke the membrane with a
quick jabbing motion, penetrating only beyond the membrane.
As soon as the tip is inside the egg, draw back the needle
quickly and move to the next egg. The injection time for each
egg should take less than a second, being inside the egg approx-
imately half a second (see Note 16).
5. When the injection is over, gently shake the plate to release the
eggs from the mesh. Swirl the plate gently to bring the eggs to
the center of the plate and pour the injected eggs into a new
50-ml plate. Store in the 18°C incubator for 3–6 h before sort-
ing fertilized eggs into 6% Ficoll/0.1× MMR.
6. Three to six hours after injection, the eggs will have reached
the 4-cell stage. Sort out fertilized eggs that have evenly divid-
ing cells. Place them in a new plate containing 6% Ficoll/0.1×
MMR. Incubate fertilized eggs at 18°C overnight (10–16 h)
(see Note 17).
7. After 10–16 h, sort out good embryos into plates filled with
sterilized 0.1× MMR/10 μg/ml gentamicin (prepare the solu-
tion using sterilized MilliQ water). Sort the embryos once
again in the afternoon and change the plate filled with steril-
ized 0.1× MMR/10 μg/ml gentamicin. Store in the 18°C
incubator overnight.
8. Sort out good embryos into new plates filled with sterilized
0.1× MMR and continue daily water changes with 0.1× MMR
until day 7, when they are ready for sorting under the dissect-
ing microscope and UV illumination.
3.2. Interphase Egg 1. Prime ten female frogs (X. laevis) 3–5 days prior to HCG injec-
Extract Preparation tion by injecting 100 U of PMSG into the dorsal lymph sac
(Fig. 2a). Maintain at room temperature.
2. The evening before the extract preparation, inject each frog
with 500–700 U of HCG (Chorulon) and place two frogs per
tank containing frog water. Place the frogs in the 15–18°C
incubator overnight (see Note 18).
3. On the day of the procedure remove frogs from the incubator

and place each frog in a tank filled with 1× egg laying solution
(at frog-room-temperature, 20°C) (see Note 19).
4. Gently collect eggs from each tank using a collecting pipette
and place them into 50-ml conical tubes. Examine a sample of
eggs from each tank under the dissecting microscope to deter-
mine if they are healthy (i.e. they have a good jelly coat and a
clear maturation spot; in contrast, unhealthy eggs are large and
white, overly speckled or stringy). There should be a total of
100 ml or more eggs collected into several tubes. Pull out any
dead (white) or unevenly pigmented eggs (see Note 20).
5. Divide eggs between several 50-ml conical tubes to have
approximately 20 ml of eggs in each tube. Pour off as much
liquid as possible. Wash eggs with 1× MMR three times or
more, until the solution is clear. Remove as much liquid as pos-
sible using a collecting pipette.
6. Fill up the conical tube with 2% cysteine solution (to the 45 ml
line). Put the cap on and gently invert tubes repeatedly to
remove the jelly coat from the eggs. Observe the eggs as they
fall to the bottom of the tube. As their jelly coats are released,
the eggs become more compact at the bottom of the conical
tube. For instance, if the initial volume of eggs was 20 ml, the
volume will drop to ~10 ml after proper dejellying (Fig. 2b).
This process should take about 3–5 min depending on the thick-
ness of the jelly coat and the freshness of the 2% cysteine solu-
tion. As soon as the eggs become compact and their jelly coats
are released, pour off the cysteine and wash eggs with XB buffer
to remove the cysteine (wash with 35 ml of XB buffer five times
or more, until the solution is clear). Use a transfer pipette to
remove dead eggs from the mix (dead eggs are typically white,
larger than healthy ones, and tend to appear on the top).
7. Wash twice with 25 ml of protease inhibitor buffer in CSF-XB
(10 μg/ml, a 1:1,000 dilution) (see Note 21).
8. Transfer collected eggs into 14-ml clear tubes (JS 13.1 rotor
holds six tubes), using a wide-bore transfer pipette. Transfer
equal amounts of eggs to each tube.
9. Remove as much CSF-XB+ protease inhibitor buffer as possi-
ble from each tube. Add 1 ml Versilube F-50.
10. Spin the tubes containing the dejellied eggs and Versilube in
the JS-13.1 rotor until the rotor reaches 150 × g (1,000 rpm),
followed by 30 s at 600 × g (2,000 rpm). Eggs will be packed
after this spin but not broken (Fig. 2c). The Versilube will have
replaced the CSF-XB between the eggs. An inverted meniscus
between the Versilube and displaced CSF-XB should be clearly
visible. Remove the excess CSF-XB and Versilube and balance
the tubes (by transferring the eggs with a transfer pipette).
11. Spin the tubes for 10 min at 16,000 × g (10,000 rpm) at 4°C in
the JS-13.1 swinging bucket rotor to crush the eggs. The
crushed eggs will separate into three layers (Fig. 2d):
Top layer A thick yellow lipid layer which can gently be

removed with a cotton swab (see Note 22)
Middle layer The desired cytoplasmic layer which appears
gray; occasionally there is a second darker
cytoplasmic layer
Bottom layer A black layer consisting of unbroken eggs
and membranes
12. Collect the cytoplasmic layer by inserting an 18-gauge needle

attached to a 2-ml syringe down the side of the tube, allowing
the tip of the needle to be visible against the wall of the tube.
Aiming for the middle layer, withdraw the cytoplasm slowly
into the syringe and avoid mixing the extracted solution with
other layers. Transfer cytoplasm to a fresh 14-ml tube on ice.
Combine the cytoplasm from the two 14-ml tubes if necessary.
Estimate the approximate volume of the cytoplasm in the tube
and record it.
13. Add protease inhibitors, leupeptin and PepstatinA, to each
tube for a final (10 μg/ml) (do a 1:1,000 dilution of stock
solution) (see Note 23).
14. Spin the cytoplasm in the JS-13.1 swinging bucket rotor at
16,000 × g (10,000 rpm) for 10 min for further separation of
the cytoplasm (Fig. 2e).
15. Collect the cytoplasm as before. Expect to collect approxi-
mately 1.0 ml of cytoplasm from the initial 40 ml of eggs.
16. Measure the volume of the cytoplasm and add 1/20th volume
of Energy Mix solution. Transfer the cytoplasm to thin-walled,
polyallomer, 5-ml Beckman ultracentrifuge tubes (Beckman #
326819).
Add 1 M CaCl2 to each tube for a final concentration of
0.4 mM (1 μl/2.5 ml of cytosol) and incubate at room tem-
perature for 15 min (see Note 24).
Spin the cytoplasm in the Beckman SW55Ti swinging
bucket rotor at 270,000 × g (47,000 rpm) for 3 h at 4°C.
17. The cytoplasm will separate into four layers (Fig. 2f):
Top layer The yellow lipid layer. This should be minimal

at this step
Second layer The cytosol, a clear golden color
Third layer Membranes and mitochondria
Bottom Glycogen and ribosomes
Gently remove the lipid layer with a cotton swab. Extract the
cytosolic layer by inserting an 18-gauge needle attached to a
1-ml syringe down the side of the tube into the cytosol. Pull
on the syringe with even, gentle pressure so that the layers do
not mix.
18. Transfer the cytoplasmic layer to a fresh ultracentrifuge tube
and spin again at 270,000 × g (47,000 rpm) for 45 min at 4°C
(see Note 25).
19. Carefully remove the cytosolic layer with an 18-gauge needle
attached to a 1-ml syringe. If the procedure is performed prop-
erly, the layers should not be as evident as before and the cyto-
sol should have a clear golden color. If the cytosolic layer is
cloudy, spin in a microfuge for 15–30 min at 4°C.
20. A typical yield for this prep, for an initial 100 ml of eggs, is
1–2 ml of high-speed cytosol.
21. Dispense into 20-μl aliquots on ice.
22. Flash-freeze in liquid nitrogen and store at −80°C.
3.3. Sperm Nuclei 1. Choose six mature males showing a distinguishable dark patch
Preparation on their hand. Inject 100 U of HCG into the dorsal lymph sac
2–5 days before the procedure (see Fig. 2). Incubate the frogs
at frog-room-temperature.
2. To isolate testes, anesthetize the sexually mature males in cold
1% tricaine (ethyl-3-aminobenzoate methanesulfonate salt) for
10–15 min. Verify that they are properly anesthesized by pinch-
ing their toes with tweezers or by flipping them onto their back
(see Note 26).
3. Rinse one male at a time with water. Decapitate and pith the
male using a paper clip. Open the abdomen by cutting the skin
in the midline, followed by the abdominal wall. The addition
of two transverse cuts at the base of the abdominal wall (hypo-
gastric area) facilitates the isolation of testes. Isolate both testes
from the frog using forceps and a pair of dissecting scissors.
Avoid collecting the fat pad attached to the upper pole of the
testes during isolation. Avoid damaging the testes.
4. Rinse the testes quickly with ice-cold 1× MMR and store in a
50-ml conical tube filled with ice-cold 1× NPB. When all testes
are collected in the 50-ml conical tube, invert the tube several
times. Wash the testes two or more times with ice-cold 1×
NPB. Fill the 50-ml conical tube with fresh ice-cold 1× NPB
and maintain it on ice. Invert the tube every 5 min.
5. Place one testis at a time in a Petri dish filled with ~5 ml ice-
cold NPB, mounted on a cold surface to keep the solution
cold. Under the dissecting microscope, use #5 forceps to clean
Exploring the Variety of Random
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While the guilty mistress beheld with such spite and vexation the
departure of her youth, the virtuous wife experienced neither pain
nor regret at growing old. It is the privilege of honest women to
accept the laws of our common destiny without a murmur, and not to
attempt a foolish struggle against nature in the hope of repairing the
irreparable ravages of years. The Marquise loaded her face,
withered by anxieties, with rouge and powder, and exhausted all the
science of a desperate coquetry in the effort to keep up an illusion.
Marie Leczinska, on the contrary, did not entertain for a moment the
thought of rejuvenating herself. Casanova, who was present at one
of her dinners at Fontainebleau, represents her as “without rouge,
simply dressed, her head covered with a large cap, old-looking and
devout in aspect.” This wholly Christian simplicity was not without its
charm. The Queen possessed not merely goodness but wit, and her
qualities were reflected on her spiritual countenance without the
least pettiness, venerable with no touch of moroseness. While Louis
XV. and his mistresses were so sad, so disillusionized, so
disenchanted with everything, though surrounded by all their
voluptuous pleasures, Marie Leczinska never uttered a complaint.
Gaiety was in reality the basis of her character, not that factitious,
turbulent, ephemeral gaiety which vice knows for a moment, but that
soft, continuous, unaffected, equable gaiety imparted by a serene
disposition and a conscience in repose.
What an expression of soundness, of moral wellbeing! What
patience with life, what sympathetic serenity! The Queen is
interested in many things; she is fond of honest amusements. Unlike
Louis XV., who is bored by everything, she has a taste for music; she
paints a little, she embroiders, she plays the guitar, the hurdy-gurdy,
the harpsichord; she willingly takes part in games of chance.
President Hénault introduces us into the cabinet, whither she
withdraws after having dined alone in public, in accordance with the
formalities of etiquette: “Here,” he says, “we are in another climate;
this is no longer the Queen, but a private person. Here one finds
work of all descriptions, tapestry, arts of every sort, and while she is
working she kindly tells us what she has been reading; she mentions
the parts that have impressed her and appreciates them.”
Look at Latour’s pastel, so admirably described by Sainte-Beuve.
“It is a half-length portrait of the Queen. She holds a closed fan in
one hand; she turns toward the spectator like some one who is
thinking, and who is going to say something arch, some innocent
piece of slyness. Her hair is slightly powdered; on her head she
wears a point of black lace, a sort of little fichu called a fanchonnette;
a mantelet of pale blue silk, with puffings or ribbons of grayish white,
the shades are so blended that they lose themselves in each other.
A tranquil harmony pervades all the tones. The lips delicate,
somewhat thin, turning up at the angles; the eye small and brilliant;
the nose a trifle saucy,—everything in this countenance breathes
gentleness, subtlety, archness. If you know neither her rank nor her
name, you will say that this middle-aged person can certainly make a
sound and appropriate repartee; that she has the grain of salt
without bitterness.”
How many times, at Versailles, I have stopped for a while in the
Queen’s bedchamber,[57] in that chamber which was occupied by
Marie Leczinska from December 1, 1725, the day of her arrival at the
palace of Louis XIV., until June 24, 1768, the day of her death! At the
back of the former alcove, on the right, over a door which led to the
small apartments of the Queen,[58] now hangs Nattier’s fine portrait
of Marie Leczinska. The wife of Louis XV. is sitting down, dressed in
a red gown bordered with fur, her arm leaning on a pier-table, on
which lie the crown, the royal mantle, and a New Testament. There is
nothing studied, nothing theatrical, in either the pose, the
countenance, or the costume. It is a blending of kindliness and
dignity. It is a queen, but a Christian queen.
After the pencil, the pen; after Nattier, Madame du Deffand. Listen
to the famous Marquise, ordinarily so sarcastic:—
“Thémire has much wit, a sensitive heart, a kindly disposition, an
interesting face. Her education has imprinted in her soul a piety so
veritable that it has become a sentiment, and one which serves her
to regulate all others. Thémire loves God, and next to him all that is
lovable; she knows how to bring solid matters and agreeable ones
into harmony. She occupies herself with each in turn, and sometimes
combines them. Her virtues have, so to say, the germ and pungency
of passions. To admirable purity of manners she joins extreme
sensibility; to the greatest modesty a desire to please which would
by itself achieve its object. Her discernment makes her penetrate all
caprices and understand all follies; her goodness and charity make
her endure them without impatience, and rarely permit her to laugh
at them.... The respect she inspires is based rather on her virtues
than her dignity. One has entire freedom of mind when with her; one
owes it to the penetration and delicacy of hers. She understands so
promptly and so subtly that it is easy to communicate to her
whatever ideas one desires, without infringing the circumspection
demanded by her rank. One forgets, on seeing Thémire, that there
can be other grandeurs, other elevations, than those of her
sentiments; one almost yields to the illusion that there is no interval
between her and us than that of the superiority of her merits; but a
fatal awakening acquaints us that this Thémire, so perfect, so
amiable, is the Queen.”
No one was a more faithful friend than Marie Leczinska. The little
circle amidst which she lived displayed as much affection as respect
for her. After supper she went almost every evening to the apartment
of the Duchess de Luynes, her lady of honor. There she met,
besides the Duke and Duchess, Cardinal de Luynes, the Duke and
Duchess de Chevreuse, and President Hénault. This was a time of
recreation and pleasant talk. The learned president shone there by
his wit. One day he offered the Queen the manuscript of his Abrégé
chronologique. She returned it with the following words: “I think that
M. Hénault, who says so many things in so few words, can hardly
like the language of women who talk so much to say so little.” In lieu
of signature, she had written: Devinez qui (Guess who). The gallant
author replied at once:—
“Ces mots tracés par une main divine
Ne peuvent me causer que trouble et qu’embarras.
C’est trop oser si mon cœur les devine;
C’est être ingrat que ne deviner pas.”[59]
Another time, Fontenelle, then ninety-two years old, had
addressed these verses to the President:—
“Il fallait n’être vieux qu’à Sparte,
Disent les anciens écrits.
Grand Dieu! combien je m’en écarte,
Moi qui suis si vieux dans Paris.
O Sparte! ô Sparte! hélas! qu’êtes-vous devenue?
Vous saviez tout le prix d’une tête chenue.
“Plus dans la canicule on était bien fourré,
Plus l’oreille était dure et l’œil mal éclairé,
Plus on déraisonnait dans sa triste famille,
Plus on épiloguait sur la moindre vétille,
Plus on avait de goutte et d’autre béatille,
Plus on avait perdu de dents de leur bon gré,
Plus on marchait courbé sur sa grosse béquille,
Plus on était, enfin, digne d’être enterré,
Et plus dans ses remparts on était honoré.
“O Sparte! ô Sparte! hélas! qu’êtes-vous devenue?
Vous saviez tout le prix d’une tête chenue.”[60]
After reading these verses, the Queen wrote to President Hénault:

“Say to Fontenelle that a head like his ought to find Sparta
everywhere.” The old man, very much flattered, responded by the
following quatrain:—
“Les ans accumulés me poussent trop à bout.
Je ne puis plus, hélas! trouver Sparte partout,
Mais vous, le modèle des reines,
Vous devez bien trouver partout Athènes.”[61]
The kindly, affectionate character of Marie Leczinska is fully

displayed in the simple and friendly letters she addressed to the
Duchess of Luynes, her lady of honor. We cite several of them taken
at hazard:—
“December 22, 1750.—Nothing could give me a greater pleasure
than your letter, if I did not expect one still more sensible in four
weeks, that of seeing you. Nevertheless, it is true, that to give me
news of yourself sometimes, if you can do so without injuring
yourself, would help to alleviate a time which already seems very
long to me. All I ask of you is not to be thankful for my friendship; it is
wholly due to you. Your letter affected me to tears. Yes, God will
preserve you as long as I live; I ask it of Him with all my heart. When
I write to M. de Luynes, I say: ‘I embrace Madame de Luynes,’ but
since it is to you for him, I think it more honest to beg you to take the
trouble for me. And Monseigneur, what would he like? I think it would
be better to enclose all in the benediction I ask for him.”
The Duke de Luynes had once sent the Queen a casket as a
New-Year’s gift. Marie Leczinska thanked him in the following note,
dated January 1, 1751: “It is useless to say the casket is charming,
new in style, in a word, nothing so pretty in the world; one knows all
that. But what one doesn’t know is that I am like a child with a
plaything that pleases it. It pleases me with the same candor, except
that the gratitude proceeds from a person who knows the world a
little, and even at her own expense, and whom God has granted the
grace of having amiable and estimable friends wholly corrupt though
she is.”
Among other things the casket contained a pair of spectacles of
which the good Queen’s eyes stood in need. “Here I am gay for the
whole day from Madame de Luynes’ good-night,” she wrote to the
Duke, January 2, 1751. “Do you know what I was doing when I
received Monseigneur’s letter? I was with ... guess who? ... my fine
new spectacles (les beaux yeux de ma cassette). Never did l’Avare
love his own so much. I am hurrying to get to High Mass. I embrace
Madame de Luynes, I bow before Monseigneur, and I wish you
good-day.”
The Duchess’s shortest absences seemed like an eternity to
Marie Leczinska. At such times she wrote letter on letter to her lady
of honor, saying that long correspondences are the delights of
friendship. Here is a letter which shows what a tender friend the
Queen was. On receiving this heartfelt epistle, the Duchess de
Luynes must have been profoundly affected:—
“January 23, 1751.—Do you know what pleasure I gave myself
last evening? I went to surprise M. de Luynes in his apartment; I
found him just as he had finished his supper with Monseigneur (the
Bishop of Bayeux), in his pretty little room. I cannot tell you what joy I
felt in seeing your apartment again; I rested there a moment in order
to preserve it, for, not finding you there yet, I began to be afraid of
what might succeed it. Pleasures which are only imaginary need to
be taken care of. I impatiently await the real ones.”
To great goodness Marie Leczinska joined solid information. She
knew six languages,—Polish, French, Italian, German, Swedish,
Latin. Men of letters were struck by the shrewdness of her judgments
on the things of the mind. Several of her maxims have been
preserved, which attest a lofty soul and a profound knowledge of the
human heart. Here are some of them: “We ought not to reflect more
on the faults of others than will suffice to preserve ourselves from
them.—Human wisdom teaches us to conceal our pride; religion
alone destroys it.—To live peaceably in society, we must open our
eyes to the qualities which please us, and shut them on the follies
and caprices which shock us.—The women who pique themselves
most on knowing what it is allowable for them to be ignorant of are
those who care least about instructing themselves concerning what it
is shameful not to know.—Many princes having regretted, when
dying, that they had made war, we never see any who repented of
having loved peace.—Good kings are slaves, and their people are
free.—The only thing which can make amends for the slavery of the
throne is the pleasure of doing some good.—In politics, as in morals,
the shortest way to make men happy is to endeavor to make them
virtuous.”
The sovereign who expressed such thoughts as these was not an
ordinary woman. She surpasses all the favorites of her husband, not
merely in heart and virtue, but also in intelligence, knowledge, and
wit.
XIV
MARIE LECZINSKA AND HER DAUGHTERS
M arie Leczinska was a tender mother. She surrounded her

daughters and her son with the most devoted cares, and knew
how to inspire them with Christian sentiments. M. Michelet, who,
in his latest works, tried to sully whatever he touched, has tried in
vain to cast odious ridicule on the daughters of Louis XV. In spite of
his venomous insinuations, his calumnious influence, he has been
unable to extinguish the aureole of purity surrounding the brows of
these virtuous princesses. The truth may be found in the excellent
work of M. Édouard de Barthélemy, an impartial judge, a critic full of
sagacity.[62] A curious book, recently published by M. Honoré
Bonhomme,[63] has also avenged the memory of the daughters of
Louis XV. against attacks which the most bitter adversaries of the
monarchy and the most violent of pamphleteers had not permitted
themselves.
All of the daughters of the King, with the exception of Madame
Adelaide, spent their childhood at the Abbey of Fontevrault. Cardinal
Fleury thought the presence of the little princesses at Versailles
entailed too much expense, and Louis XV., yielding to the
suggestions of his parsimonious minister, regretfully determined on
separating himself from his children. Adelaide alone, by dint of
prayers and supplications, was able to escape the abbey. On
returning from Mass, she threw herself at her father’s feet, and,
although only seven years old, succeeded in gaining her cause. The
King wept a little, says Barbier, and promised her that she should not
go away.
It is easy to comprehend how much the good Queen must have
suffered from this parting with her daughters. She wrote to the
Duchess de Luynes, October 12, 1747: “The King surprised me by
showing me the portraits of my daughters from Fontevrault. I did not
know they had been painted. The two eldest are really beautiful, but I
have never seen anything so agreeable as the little one. She has an
affecting expression, very remote from sadness. I have never seen
anything so singular; she is touching, sweet, spiritual. If you find my
letter too long, make allowances for the tenderness of a mother and
the confidence of a friend.”
The six daughters of Louis XV. were born: the twins, Elisabeth
and Henriette in 1727; Adelaide in 1732; Victoire in 1733; Sophie in
1734; Louise, the future Carmelite, in 1737.
The three princesses of whom the Queen speaks in the letter we
have just quoted, and who were still at Fontevrault, were Victoire,
Sophie, and Louise. The twins, Elisabeth and Henriette, had quitted
the convent in 1739, and the former had soon afterwards married the
Infant Don Philip, son of Philip V., King of Spain. Thereafter she is
designated as Madame Infanta. The six sisters were all spoken of as
Mesdames de France. Nevertheless, there was but one of them who
married. When she took her departure for Spain, at the age of twelve
years (August 31, 1739), the twin sisters exchanged heart-rending
farewells. They could not resign themselves to separation. “’Tis
forever!” they cried, their voices broken by sobs. Louis XV.
accompanied his daughter as far as Plessis-Picquet. The Duke de
Luynes relates that while on the road he gave his dear child most
pathetic advice concerning the conduct she should observe in her
new country, where, said he, her mild temper would infallibly win all
hearts. He spoke to her with so much affection and tenderness that
all who were in the carriage were melted to tears.
In 1748, the husband of Madame Infanta obtained the sovereignty
of Parma, Piacenza, and Guastalla. Before going to their new
dominions with her husband, the daughter of Louis XV. came to see
her parents at Versailles. This was a delightful moment for the royal
family. Princes and princesses made few journeys in the eighteenth
century. What joy to embrace a father, mother, brother, sisters, who
had never expected to see one again! Marie Leczinska returned
thanks to heaven. The little girl of twelve, who had left Versailles,
returned thither a young woman in all the brilliancy of her twenty-
second year. The Dauphin was beside himself with joy. In the first
moment he embraced every one he saw, even the lady’s maids
(December, 1748). Sophie and Louise were still at the convent of
Fontevrault, but Henriette, Adelaide, and Victoire were at Versailles.
Their sister’s arrival was an extreme happiness for them. Madame
Infanta, so delighted to be once more with her family, had not
courage to leave them. Months passed without her being able to
decide on quitting Versailles, where her filial and sisterly heart
experienced emotions so sweet. Nevertheless, it was necessary to
be resigned. The dreaded moment arrived in October, 1749. The
farewells must be spoken. It cost Henriette so much to part with her
beloved sister that she fainted several times. The Dauphin was in
tears, and Louis XV., who loved his daughters most profoundly,
showed by his grief all the strength of his paternal tenderness.
Madame Infanta returned to Versailles some years later, but at
that time the joy of her return was not untroubled. The Princess no
longer found her twin sister, that dear Henriette whom she regarded,
so to say, as the half of her soul.
Henriette had just died at the age of twenty-four (February 10,
1752). This young girl, as unhappy as sympathetic, was certainly
one of the most touching figures in the feminine gallery of Versailles.
M. Honoré Bonhomme has made an exquisite portrait of her, from
both the physical and the moral point of view: “Of a sickly
constitution, Madame Henriette had that ivory whiteness of
complexion peculiar to the daughters of the North, and which her
mother, Polish in blood and race, seemed to have transmitted to her
along with life. Delicate, tall, and slender, there was something
dreamy and inspired in her person. Her mild, pure features,
aristocratic in their outline, charmed and yet inspired respect; her
smile was melancholy, and her whole appearance, in which gloom
seemed constantly warring against brightness, bore the impress of
fatality. It was because she carried in her heart the secret of her
destiny. Like pale Ophelia, she was to die while gathering flowers,
and like Myrto, the young Tarentine of André Chenier, she was never
to cross the threshold of the spouse. For the rest, inwardly animated
by the sacred fire, enamored of great things, she possessed all
subtleties of the mind as well as all delicacies of the heart. Looking
into her great, dreamy eyes, which seemed to reflect the dormant
limpidity of deep lakes, one divined what abysses of tenderness and
devotion were hidden underneath, and felt a presentiment that her
first love would also be her last, that she would die there where her
soul had fixed itself.”
That, in fact, is what happened. Madame Henriette had conceived
for the young Duke de Chartres, son of the Duke d’Orléans, an
affection which was returned. The Marquis d’Argenson wrote,
November 30, 1739: “A secret effort is being made to bring about a
marriage between the Duke de Chartres and Madame seconde [so
Madame Henriette was called; her twin sister, Madame Infanta, was
known as Madame première], and it is believed that the King is
determined on it and gradually working toward it. Nothing could be
more conformable to pacificatory views, for Europe would plainly see
from this that the King was disposed to substitute the Orleans branch
to the Dauphin, rather than the Spanish one.”
To understand this phrase, it is necessary to recall that the
Dauphin was not yet married, and that people often wondered what
would happen if this only son of Louis XV. should die without male
posterity. Many thought that in such a case the King, in spite of the
renunciations of the treaty of Utrecht, would take his heir from the
Spanish Bourbons, and not from the Orleans branch. D’Argenson
was in favor of the latter branch. Cardinal Fleury, on the contrary,
pursued it with hostility, as if he had an insight of the future. The old
minister prevailed so far, that the King, who had nevertheless a real
liking for the Duke de Chartres, an amiable and estimable young
prince, would not give his consent to the projected marriage. One
day the Duke was riding beside the King. “Sire,” said he, “I had a
great hope. Your Majesty had not taken it from my father.... I could
have contributed to the happiness of Madame Henriette, who would
have remained in France with Your Majesty. May I still be allowed to
hope?” The King inclined toward the Prince and sadly pressed his
hand. This beautiful dream of love, so quickly faded, must be
renounced. Three years later, the Duke de Chartres espoused the
daughter of the Prince de Bourbon-Conté. Madame Henriette had
the courage to conceal her immense sorrow. She was present, death
in her soul, a smile on her lips, at the marriage of the man she loved
(December 9, 1743). From that day she felt herself heart-stricken,
and her last days were merely an immolation. Prince Nattier has
represented the Princess under the double emblems of Fire and
Meditation. She is leaning against a tripod on which half-consumed
torches are smoking. These torches are like the image of the nearly
extinguished flame of the Prince to whom the young girl would
willingly have given her faith. She never uttered a complaint, a
murmur. Calm, grave, recollected, she meditated and she prayed.
The stay of her twin sister at Versailles was like a break in the
darkness of her night. But when this dear companion of her infancy
departed, all the wounds of her tender and loyal heart reopened.
The arrival of her three younger sisters, Victoire, Sophie, and
Louise, who left the convent of Fontevrault at the close of the year
1750, did not console her. Having sacrificed her own happiness, she
desired that at least the Duke de Chartres might be happy. But it was
not so. The Duke had married a woman whose conduct was said to
be anything but exemplary. He could not, then, forget that tender,
that virtuous Henriette who seemed to him the image of sadness.
The Princess wept silently in her oratory, and offered her sufferings
to God. Earth was not worthy of her.
There are characters which can only expand in a better world.
Sorrow had undermined the constitution of Madame Henriette. She
died February 10, 1752. “Ah! my sister! my dear sister!” were her last
words. She died as she had lived: while loving. “Sad sport of fate,”
says M. Honoré Bonhomme, “poor saintly girl, virgin and martyr, who
spent nine whole years in climbing, step after step, the Calvary
where she yielded up her soul.”
After relating this death, the Duke de Luynes adds: “No one can
express the sadness into which the King is plunged. The Queen is
much afflicted, and also the Dauphin, Madame the Dauphiness, and
Mesdames. Madame Adelaide does not weep, but silent griefs are
usually the longest. Madame Henriette was much beloved. Her mild
character, without ill temper and even without will, rendered her
extremely complaisant toward the Dauphin, the Dauphiness, and the
ladies, her sisters.”
On receiving tidings of this mournful death, Madame Infanta wrote
her father a most touching letter. She said she wished to come and
mingle her tears with those of her family. She arrived in France in
September, 1752, and remained with her father for a year.
Madame Infanta was not happy. She did not greatly esteem her
husband, and that Prince cut a rather sorry figure in the little
sovereignty of Parma and Piacenza. He had neither money nor
prestige; and his wife, who was very intelligent, his wife, of whom
Bernis said that she would make a good minister of foreign affairs,
was constantly dreaming of some more considerable establishment
for him. She thought by turns of exchanging the Duchy of Parma for
Tuscany, or acquisitions in Flanders, Lorraine, or even Corsica. She
fancied that, thanks to her father’s affection and the territorial
changes in Europe, she would end by obtaining something. The
Marquis d’Argenson, who had not much sympathy for her, wrote,
September 27, 1753: “It is to be hoped she will never come back to
France. Is it just that the State should suffer because she was
married so badly? Along with her go a great quantity of chariots
loaded with all sorts of things that the King has given her.”
Madame Infanta returned to France a third time, but only to die
there. She arrived at the château of Choisy, September 3, 1757. To
credit M. Michelet, it was she alone who brought about the Seven
Years’ War. But there is no foundation for this assertion of the great
writer who, toward the close of his life, created what one might call
the school of imaginative history. At the time when she reappeared
at court, Madame Infanta was glowing with freshness, brilliancy, and
health. No one could have foreseen that her death was so near at
hand. One of her last letters was addressed to her son Ferdinand,
whom she had left at Parma. It commenced as follows:—
“Life is uncertain, my son, and my character is too sincere for me
either to vaunt or even to affect perfect indifference as to the length
of mine; but I feel that the wish to see you, to leave you worthy of the
name you bear in the world, such, in fine, as I desire you, is one of
the ties that attach me most to life, and one of the reasons, perhaps,
which will most abridge mine by the continual torments caused me
by this desire and the fear of not obtaining it. It will be a great
consolation to be able to leave you an avowal of my sentiments if I
die before you are in a condition to read it. If I live, it will serve me as
a plan whereon to form you; and in either case, it will always be to
you a proof of my tenderness and of my care for your welfare at an
age when many people do not yet think of it.”
Not many days after writing this letter, Madame Infanta was
attacked by small-pox, and died December 6, 1759. The twins, who
had loved each other so tenderly, both died prematurely. Madame
Henriette had died at the age of twenty-four, Madame Infanta at
thirty-two. She was buried at Saint Denis, close to her sister, so that
their union lasted even in the tomb.
Marie Leczinska’s heart was broken with grief. But instead of
murmuring against Providence, she bent filially beneath the hand of
God who smote her. Her five remaining children, the Dauphin,
Adelaide, Victoire, Sophie, and Louise, showed her a profound
affection. Never was a mother better loved. Louis XV. took pleasure
in the society of his daughters. As a father, he had that sort of
citizenlike good-nature which is unhappily rare among princes.
Mesdames lodged underneath their father, in the former apartment
of Madame de Montespan. Madame Adelaide occupied a chamber
which communicated by a private staircase with that of her father.
“Often,” relates Madame Campan in her Memoirs, “he brought and
drank coffee there which he had made himself. Madame Adelaide
pulled a bell-rope, which announced the King’s visit to Madame
Victoire. On rising to go to her sister, Madame Victoire rang for
Madame Sophie, who in her turn rang for Madame Louise.”
In a twinkling the four sisters were gathered around their father. At
six in the evening, at the unbooting of the King after the chase, as
people said in those days, the princesses came to pay a visit to
Louis XV., but this time with a certain etiquette. “The princesses,”
says Madame Campan again, “put on an enormous hoop which
supported a skirt braided with gold and embroideries. They fastened
a long train to their waist, and hid the negligence of the rest of their
habiliments by a large cape of black taffeta, which covered them up
to the chin. Knights of honor, ladies, pages, equerries, ushers,
carrying large torches, accompanied them to the King. In an instant
the whole palace, usually solitary, was in movement; the King kissed
each princess on her forehead.” In reality, he found more true
happiness in the virtuous intimacy of his daughters than in the circle
of his courtiers and the arms of his favorites. There were moments
when people believed that in growing old the debauchee would
become wise. “The King,” wrote D’Argenson, “seems to wish for no
society but that of his family, like a patriarch and a good man.”
Marie Leczinska felt thankful to her husband for the affection he
had for his daughters. The relations of Mesdames with their mother
were full of confidence, sweetness, and gaiety. They liked to enter
those little apartments of the Queen, where Marie Leczinska forgot
the splendor of the throne to live modestly as a good mother. The
little apartments[64] comprised three rooms: a salon, a bathroom,
and a studio for painting. Madame the Countess d’Armaillé, whose
graceful and solid work we have so often had occasion to quote, has
given a charming description of these three rooms in which Marie
Leczinska spent the greater portion of her time. “Is it not true,” she
says, “that one may divine the character and tastes of a woman by
merely inspecting the sanctuary of her private life, or, to speak more
simply, that place in the dwelling where she habitually prefers to
stay? It matters little whether this room be a garret or a drawing-
room. Nothing is so intimate as certain interior arrangements;
nothing tells the story of a woman better than the way in which she
orders the room she inhabits. In the little apartments of the Queen
one found everything which makes the charm of a peaceful
existence. Here, pieces of work begun for the poor, or for churches,
a whole piece of furniture embroidered by her hand; there, an open
harpsichord with Moncrif’s cantatas, Rameau’s operettas, Polish
hymns; further away a drawing-table, a spinning-wheel provided with
its distaff, frames for embroidering and weaving, a small printing-
press; then flowers, paintings, portraits of children, miniatures. On a
console, a vase offered by Marshal de Nangis, a manuscript given
by Cardinal de Fleury, a porcelain pagoda with verses by Madame
de Boufflers; in an embrasure of the window a cabinet containing the
Queen’s favorite books, with some verses by the Duchess de
Luynes; everywhere souvenirs of friendship, of maternal tenderness,
of useful or agreeable occupations.” It was there that, surrounded by
her children, the virtuous Queen tasted the joys of the heart, those
joys imparted only by a good conscience, and which the mistresses
for whom Louis XV. deserted her had never known.
XV
THE DAUPHINESS MARIE JOSÈPHE OF SAXONY
M arie Leczinska was not less happy in her son than in her
daughters. The bad examples of the court had not spoiled the
upright and honest nature of the Dauphin. As is said by Baron
de Gleichen in his Memoirs, the piety of the young prince was
enlightened, and his policy foresaw the dangers of irreligion. As son
and father, as brother and husband, he never ceased to display the
qualities of a good and virtuous heart. He had deeply mourned his
first wife, that sympathetic Spanish Infanta, who died in 1746, when
hardly twenty years old. Reasons of State demanded that, in spite of
his great sorrow, he should promptly contract a second marriage.
Louis XV. selected for his son a princess of the house of Saxony,
after Austria and Prussia the most powerful of the Empire. He
intended thus to consolidate his German alliances. Marshal Saxe,
natural son of Augustus II., King of Saxony, Elector of Poland, and of
the beautiful Countess Aurora of Königsmark, was the principal
agent of the negotiation which was to form a pact of union between
his new country and his old one. A learned Saxon diplomatist, now in
the service of Austria, Count Vitzthum, published some years ago an
excellent work, based on unpublished documents and letters in the
archives of Dresden, on the Marshal and the princess who espoused
the Dauphin.
Marie Josèphe of Saxony, daughter of Augustus III., was at this
time fifteen years old. She was an agreeable young person, with
large blue eyes that were at once keen and gentle. Her countenance
was intelligent, her character excellent, her education complete.
Marshal Saxe wrote to his brother, Augustus III.: “Sire, what shall I
say to you? I find this affair advantageous at all points for your family,
and I shall descend without regret to the empire of the shades after I
have seen it terminated; I shall have accomplished my career. I have
enjoyed the delights of this world; glory has covered me with its
benefits; nothing more remained to me but to be useful to you, and
all my destiny will have been fulfilled in a most satisfactory manner.”
Marshal Saxe wrote to the wife of Augustus III., mother of the
future Dauphiness:—
“Madame, the Most Christian King sent me word yesterday, that
he had requested Your Majesty for the hand of the Princess Marie
Josèphe for Monseigneur the Dauphin. I flatter myself that this
proposition will not displease either the Princess or Your Majesty, for,
in truth, Monseigneur the Dauphin is a very good match, and I
should like to live long enough to see our divine Princess Queen of
France. I think that would suit her very well. She has always been
my inclination, and it is long since I destined her for the crown of
France, which is a fine enough morsel, and the Prince who will some
day wear it is fine also. The Princess Josèphe will have no reason to
be bored while she is waiting for it. The kingly father-in-law is
charming; he loves his children, and from the caresses he gave the
late Dauphiness, I infer those which our Princess will have to endure.
This is word for word what the King wrote me in a letter I received
yesterday, written by his own hand from one end to the other. ‘You
will not be vexed with this marriage, my dear Marshal? Let your
Princess be sure that it depends on her alone to make our happiness
and the felicity of my people.’”
In the same letter the Marshal gave some very sensible and
prudent counsels: “I will say another word to the Princess. To
succeed here, neither hauteur nor familiarity is required; hauteur,
however, pertaining to dignity, she can more easily incline to that
side. The women of the court all have minds like diamonds, and are
wicked withal. No one will fail in respect towards her, but they will try
to entangle her in their continual quarrels, and at these she must do
nothing but laugh and amuse herself. This is what the King does;
and if anything displeases her, she must address herself directly to
the King: he will advise and conduct her very well. This confidence
will please him. He is the only person at court with whom she should
have no reserve. She should regard him as her refuge, her father,
and tell him everything, good or bad, just as it happens, without
disguising anything. With everybody else, reserve. If she does that,
he will adore her.”
The formal demand in marriage was made at Dresden, January 7,
1747, by two ambassadors, one extraordinary, the Duke de
Richelieu, the other ordinary, the Marquis des Issart. Richelieu wrote
to the Count de Loss, apropos of the future Dauphiness: “I find her
really charming; nevertheless, she is not a beauty, but she has all
the graces imaginable; a large nose, thick, fresh lips, the brightest
and most intelligent eyes in the world, and, in fine, I assure that if
there were any such at the Opera, they would soon be put up at
auction. I do not say too much to you, but I do not say so much to
others.”
Marie Josèphe of Saxony left Dresden, January 14, 1747. She
saw her betrothed for the first time between Nangis and Corbeil. The
nuptial benediction was given to the pair in the chapel of Versailles,
February 8, 1747. Four days afterward, Marshal Saxe wrote to
Augustus III.: “Sire, I shall have no difficulty in saying agreeable
things to Your Majesty about Madame the Dauphiness, and renown
will serve as my guaranty. No one could succeed better than this
Princess; she is adored by everybody, and the Queen loves her as if
she were her own child; the King is enchanted with her, and M. the
Dauphin loves her passionately. She has steered her way through all
this with all imaginable address; I could not but admire her. At fifteen,
according to what they say, there is no such thing as childhood in
this society; and, in truth, she has astonished me. Your Majesty
could hardly believe with what nobility, what presence of mind,
Madame the Dauphiness has conducted herself. M. the Dauphin
seems a schoolboy beside her.”
The married pair were installed on the ground floor, in the south
wing of the central portion of the palace, under the Queen’s
apartments. (The Dauphin’s bedroom, where the Regent died, is
now the third hall of the Marshals, No. 46 of M. Eudore Soulié’s
Notice du Musée. That of the Dauphiness is now the second hall of
the Marshals, No. 41 of the Notice.) It was in the latter chamber that,
according to usage, the ceremonial of the putting to bed took place.
In the letter we are about to quote, Marshal Saxe gives his brother
an account of this strange custom:—

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PART I MANIPULATION OF GENE EXPRESSION IN VIVO

1 Conditional Control of Gene Expression in the Mouse Retina. . . . . . . . . . . . . 3

PART II TRACING CELL FATE

7 Testing Retina Fate Commitment in Xenopus by Blastomere Deletion,

PART III IN VITRO SYSTEMS

10 In Vitro Explant Culture and Related Protocols for the Study

PART IV REGENERATION/STEM CELLS

15 Studying the Generation of Regenerated Retinal Neuron from Müller

19 Measuring Rodent Electroretinograms to Assess Retinal Function . . . . . . . . . . 265

PART VI EMERGING METHODOLOGIES

21 Use of Laser Capture Microdissection for Analysis of Retinal

TERI L. BELECKY-ADAMS • Department of Biology & Center for Regenerative

PETER HITCHCOCK • Department of Ophthalmology and Visual Sciences,

GUANG-HUA PENG • Department of Ophthalmology and Visual Sciences,

XIAN-JIE YANG • Department of Ophthalmology, Molecular Biology Institute,

Manipulation of Gene Expression In Vivo

Conditional Control of Gene Expression in the Mouse Retina

Gene targeting is a powerful tool in studying in vivo function of

Fig. 1. Schematic diagram of tissue-specific controlling of gene expression by Cre recom-

to the genome at desired sites by homologous recombination

2.1. ES Cells 1. W4 ES cells.

2.3. Electroporation 1. Electroporation apparatus.

2.4. Southern Blot 1. ES cell lysis buffer: 10 mM Tris–HCL, pH 7.5, 10 mM EDTA,

2.5. Microinjection 1. Blastocysts from pregnant female mice.

2.7. Genotyping 1. Tail digestion buffer: 10 mM Tris–HCL (pH 8.0), 25 mM

3. PCR mixture: 1 mg DNA as template, 1× PCR buffer,

3.2. Manipulation 1. Gelatinize plates: add 5 ml of gelatin solution to each 10-cm

3.3. Electroporation 1. Linearize 200–300 mg of Isl1 conditional knockout targeting

3.4. Screening for 1. Prepare 96-well plates with feeder cells.

of 96-well plate containing 35 ml of 0.05% trypsin at room

3.5. Blastocyst 1. Harvest E3.5 blastocysts from pregnant females.

2. Weigh and anesthetize the recipient female with avertin.

3.7. Determination 1. A convenient way to determine the contribution of targeted

5. Dry the DNA and dissolve in 50 ml of TE. The DNA

1. The construction of targeting vector is very important for the

event happens (15). Meanwhile the floxed sequence is also

the model is to a desired purpose. In addition, a uniform genetic

Promoter Targeted cell types References

Pax6 Distal neural retina (23)

1. Ferrara N, Carver-Moore K, Chen H, Dowd 7. Beglopoulos V, Shen J (2004) Gene-targeting

Generation of Transgenic Xenopus Using Restriction