505

Transgenic Mice in Biomedical

Research

J. Willem Voncken and Marten Hofker

University of Maastricht, Maastricht, The Netherlands

1 A Brief History of Mice 508

1.1 Mouse Genetics 508
1.2 Molecular Mouse Genetics 510
1.2.1 Brief Overview of Developments in Transgenesis 511
1.2.2 Brief Overview of Developments in Gene Targeting 512

2 Transgenesis; Basics and Technology 513

2.1 Transgene Design 513
2.1.1 Origin and Structure of the Transgene 513
2.1.2 Regulatory Elements 515
2.2 Pronuclear Injection 518
2.3 Biotechnological Procedures 518

3 Gene Targeting; Basics and Technology 521

3.1 Targeting Vector Design 521
3.2 Embryonic Stem (ES) Cells 524
3.3 Genesis of Mosaic Embryos 524
3.4 Biotechnological Procedures 526

4 Refinements to the Models 527

4.1 Fully ES Cell–derived Embryos 527
4.2 Conditional Transgenesis 529
4.3 Conditional Gene Targeting 530
4.3.1 Controlled CRE Expression 532
4.4 Knockins: Humanized Mice 533

5 Random Mutagenesis in ES Cells 536

5.1 Gene Trap Mutagenesis 537

Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Edition. Volume 14
Edited by Robert A. Meyers.
Copyright  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-30651-X
506 Transgenic Mice in Biomedical Research

5.2 Retroviruses and Transposable Elements in Random

Mutagenesis 538
5.3 Random Enu Mutation 539

6 Phenotype Analysis 539

7 Perspectives 540
7.1 Partial Transgenesis; Viral Shuttles 540
7.2 RNA Interference; Posttranscriptional Gene Silencing 541

Bibliography 542
Books and Reviews 542
Primary Literature 543
Websites (References Indicated as they Appear in the Original Text) 545

Keywords
Autologous
Derived from endogenous sequences.

Binary Model
Mouse model in which gene activity can be switched on and off.

Chimeric Mouse
Mouse derived from donor ES cells and recipient embryo, displaying coat
color mosaicism.

Chromosome Substitution
A single full-length chromosome from one inbred strain has been transferred onto the
genetic background of a second strain by repeated backcrossing.

Conditional Model
Mouse model in which a modification can be activated on command.

CRE-LoxP
Bacterio phage P1-derived recombination system: the recombinase CRE-mediated
recombination between two identical recombinase recognition sites: LoxP.

CRE-MER
Fusion of a CRE recombinase with a mutated ligand-binding domain of the
estrogen receptor.

Ectopic Expression
Expression out of place or out of time.
 Transgenic Mice in Biomedical Research 507

Embryonic Stem Cells

Pluripotent stem cells derived from preimplantation embryos.

Gene Targeting
Modification of endogenous genes through use of replacement vectors.

Gene Trapping
Process by which transcription of a marker gene is coupled to that of an endogenous
locus in which the gene trap vector is inserted and transcripts are trapped.

Heterologous
Derived from exogenous sequences.

Inbred Strain
A mouse strain bred to homozygocity for all loci by 20 brother-to-sister back crosses.

Insertional Mutagenesis
Gene modification by insertion of foreign DNA vectors.

Isogenic DNA
DNA derived from the same genetic background.

Knockin
Targeted mutation of an existing locus, mostly replacing endogenous coding
sequences with functional sequences

Pluripotent
Unlimited differentiation capacity.

Recombinant Inbred Strain

Recombinant inbred strains are derived from crosses of two inbred strains.

RNA interference
RNA-mediated posttranscriptional gene silencing.

Spatio-temporal Expression Pattern

Gene activity determined in time and location.

Tetraploid
Containing four haploid genome copies.

Transgene
A(n expression) construct that is inserted into the mouse genome.
508 Transgenic Mice in Biomedical Research

Genetically modified mouse models have become a crucial tool in present day
biomedical research. Modifying genes in mice provides an excellent approach to
unravel gene function at the molecular and cellular level, as well as its role in the
physiology and pathology of the intact organism. By doing so, numerous models
have been developed that reliably replicate diseases in humans, thereby providing
insight in disease mechanisms and paving the way toward prevention and therapy.
Crucial for these advances has been the ability to modulate gene expression at will. It
is possible to increase or decrease gene expression, or eliminate the expression of a
gene completely. These alterations can be made cell type–specific and even inducible
or reversible. Moreover, it is possible to replace mouse genes with the cognate human
gene carrying a specific mutation. This chapter will deal with the essential approaches
in transgenic mouse research and its impact on biomedical research.

1 out from the other mammals as being the

A Brief History of Mice
1.1
Mouse Genetics
The resources directed toward studying
the mouse have been exponentially grow-
ing. After the completion of the human
genome sequence, the mouse was the
second mammalian genome for which
the complete sequence was determined.
There is no mammalian system that
comes close to rival the molecular and
genetic possibilities that the mouse has
to offer to researchers. Many physiolog-
ical methods that worked fine in larger
experimental animals have been minia-
turized to make use of the genetic
approaches uniquely available to mouse
researchers. What makes the mouse
so special?
The advantages that come to mind first
are obvious: for biomedical researchers,
mammals are highly preferred model
systems, because the physiology of man
and most mammals is very similar and the
pathology of most diseases is reproduced
well in animal models. The mouse stands
smallest. Mouse husbandry is therefore
easy and economical; its requirements
for food are modest, and mice are
excellent breeders with a short generation
time, allowing around four successive
generations each year. Upon completion of
the human and mouse genome sequence,
the usefulness of the mouse as a model
animal for the human has been further
reinforced. The number of genes in
both species is around 30 000. With the
exception of about 200 genes, which
were shown unique to mouse or man,
the vast majority of genes is conserved
between mouse and man. These combined
properties make the mouse an excellent
animal model to study gene function and
extrapolate the outcome to human disease.
Second, the mouse has advantages that
may be less obvious, but which relate to the
long tradition of mouse research starting
in the early 1900s. Up until then, hobbyist
mouse keepers had already been breeding
mice for their coat color variation. By
making use of these mice, it was possible
to demonstrate that this variation followed
the laws of Mendelian inheritance. Soon, it
was also realized that there was a need for

genetically homogeneous mouse strains
and the most widely used inbred strains
such as BALB/c and C57BL6 originate
from that period. An excellent historical
overview on mouse genetics is provided by
Dr. Silver.
At present, hundreds of inbred strains
have been generated (see Sect. 6). This re-
source is the key to the success of mouse
research. Inbred strains ensure that exper-
iments can be carried out in the absence of
inter-individual genetic variation, because
mice within an inbred strain are all geneti-
cally identical to each other and homozygous
throughout the genome. Moreover, each
strain has one or more unique and, often,
well-established properties that make the
strain particularly useful for a specific area
of research. For instance, some mouse
strains appeared susceptible to cancer or
cardiovascular diseases, while others are
better suitable for behavioral research. The
genetics of such complex diseases and bio-
logical processes is very difficult to study
in man. This is mainly due to the fact
that multiple genes are involved and that
the genetic basis for susceptibility to, for
instance, cancer will vary from person to
person. In the mouse, it is possible to cross-
breed a susceptible and a resistant strain.
As a result, the susceptibility to cancer will
vary in the progeny. Genetic variation in
these mice is confined to the differences
between the two parental strains, and ev-
ery given locus will only have two different
alleles. Hence, the genetics is confined
to a maximum of two alleles per locus,
which greatly simplifies genetic analysis.
These properties allowed generating spe-
cific resources to study complex diseases,
including recombinant inbred (RI) strains
(http://jaxmice.jax.org/info/recombin-
bred.html). These RI strains combine
a specific genetic contribution of one
mouse strain with the genetic background
Transgenic Mice in Biomedical Research 509
of another strain. Along these lines, a
very powerful resource has been devel-
oped recently, consisting of a panel of
inbred mouse strains carrying only a sin-
gle chromosome from the other strain
(http://jaxmice.jax.org/library/notes/
493c.html). These chromosome substitution
strains are very similar to recombinant
inbred strains, but offer a less costly strat-
egy to move from a susceptibility locus
toward the gene proper. The principles of
mouse genetics are crucial when working
with transgenic animals, because it is vi-
tal to control the genetic background in
the experiments (see below): the interac-
tion between the transgene and the genetic
background will ultimately determine the
phenotype; for example, changing the ge-
netic background on a given genetic trait
may prevent an early death of a particular
mouse model.
While mouse genetics has a major
impact on our understanding of the mech-
anisms underlying complex diseases, a
constant influx of mice carrying single
gene mutations greatly stimulates mouse
research. These mutations have either oc-
curred spontaneously in mouse breeding
programs or originated from research on
the gene toxicity of radiation and chem-
ical compounds. The latter approach has
been rejuvenated upon the availability of
better technology to identify the causative
mutations (see below). A good example
of a spontaneous mutation is the lep-
tin gene mutation that occurred in the
C57BL6 mouse. The mutation was named
ob, after obese, because the mice have no
control over their feeding behavior and be-
come overwhelmingly obese. Hence, the
phenotype could be discovered easily. The
subsequent discovery of the causative gene
marks one of the most dramatic discover-
ies in this research field.
510 Transgenic Mice in Biomedical Research

1.2 random mutagenesis, the true break-

Molecular Mouse Genetics through for the mouse as the animal of
choice came with the accomplishment of
Despite all the possibilities for the (1) transgenesis through injecting DNA in
basic biomedical research offered by fertilized oocytes and (2) gene targeting via
‘‘traditional’’ mouse genetics and the homologous recombination in embryonic
discoveries of relevant mutations through stem cells. The latter mice are commonly

100 000
Cumulative number of publications →

10 000

1000 Transgenesis

100
Gene targeting

10

0
1980 1985 1990 1995 2000 2005

(a) Publication date →

Milestones in mouse molecular genetics

Transgenesis
Viral 1976
R.Jaenisch Germ line transmission of retroviruses
infection
J.Gordon Integration and germ line transmission of Pronuclear
transgenes injection 1981
R.Brinster
F.Costantini Expression of integrated transgene Pronuclear
E.Lacy injection 1982
R. Palmiter First phenotype of a transgenic mouse Pronuclear
R.Brinster (giant mouse; rat growth hormone) injection 1982
Gene targeting
M.Evans Establishment pluripotent ES cell cultures In vitro 1981
G.Martin
M.R.Capecchi Homologous recombination in mammalian cells In vitro 1982
K.R.Thomas Homologous recombination in ES cells 1987
D.W.Melton Germ line transmission of targeted mutations in In vivo 1989
O.Smithies ES cells

(b)
 Transgenic Mice in Biomedical Research 511

known as knockout mouse models. This
combination of technologies offers the pos-
sibility to design models with ‘‘gain of
function’’ and ‘‘loss of function,’’ which is
an ideal situation for a (mouse) geneticist
(Fig. 1a).
Notably, these technological break-
throughs were facilitated by the availability
of inbred mouse strains. Transgenesis
works most efficient when the donor eggs
are produced by first generation (F1) hy-
brids between C57Bl6 and BALB/c. In the
case of the gene-targeting approach us-
ing embryonic stem cells, it is critical to
make use of the appropriate strain com-
binations when injecting the cells into
blastocysts and propagating the embryos
in foster strains. This dependence on the
genetic background provides the ability to
choose the optimal strain and may be one
of the reasons why, mice are, as of yet,
the only mammalian species suitable for
gene targeting (see Sect. 3). The goal of
altering the gene function through trans-
genesis is to generate animal models in
which the role(s) of the gene of interest
either in normal (e.g. development) or ab-
normal biological processes (e.g. disease)
is revealed. Other important applications
in fundamental science include the study
of mammalian gene expression regula-
tion. Currently, genetic modification is
carried out in a wide range of invertebrate
and vertebrate species, including mam-
mals such as mice, rats, rabbits, and
livestock such as sheep, pigs, cattle, and
recently even primates. The choice of the
models is primarily determined by its ob-
jective (e.g. improvement of economical
value or development of pharmaceutical
proteins). Although fundamental biolog-
ical processes can be well studied in
relatively simple organisms such as the
nematode Caenorhabditis elegans, or the
fruitfly Drosophila melanogaster, for experi-
mental biomedical research the laboratory
mouse is by far the most used animal
model. Below, we will briefly outline the
emergence of the two technologies and
sketch the initial experimental strategies
that caused great excitement within the
scientific community.
1.2.1 Brief Overview of Developments
in Transgenesis
By classical definition, a transgenic organ-
ism carries extra, often foreign (i.e. from
a different organism) DNA in its genome,
called a transgene. Transgenesis literally
crosses (trans) species barriers. One of the
most obvious conceptual characteristics
is that a transgene integrates at random
in the recipient host genome, whereas
gene targeting, by homologous recombi-
nation, selectively alters an existent locus.
A number of milestone achievements,

Fig. 1 Genetically modified mouse models in biomedical science. (a) A rough estimation of
published scientific output using genetically modified mouse models. The blue line represents
reports on transgenic models and the red line on gene targeting. Note: with the advent of conditional
gene targeting, which utilizes transgenesis in conjunction with targeted mutagenesis, a strict
separation of transgenesis and gene targeting into the twentieth century is difficult. (b) Milestones in
mouse molecular genetics. The first report on germ-line transmission of integrated retroviruses (via
infection) dates back to the mid-1970s. Subsequently, pronuclear microinjection of fertilized oocytes
and subsequent proof of transgene integration, expression, and germ-line transmission, were
pioneered in different laboratories. Similarly, breakthroughs in the use of embryonic stem (ES) cell
technology are listed. Many other excellent laboratories have since contributed to refinement of
technologies and strategies; these are listed throughout this chapter (see color plate p. xxvi).
512 Transgenic Mice in Biomedical Research
which were pivotal for the development
of modern-day mouse molecular genetics,
are summarized in Fig. 1(b).
In the mid 1970, robust technology to
clone genes and manipulate genes was
still about five years away. Subsequently,
as the structure of eukaryotic genes was re-
vealed and recombinant DNA technology
developed, gene constructs could be gen-
erated comprising an eukaryotic promoter
fused to the full-length coding sequence of
the gene of interest (consisting of a DNA
copy (cDNA) of the mRNA molecule).
Generating mice with a strong promoter
driving an extra copy of the gene resulted
in mouse models with high-level expres-
sion of the gene of interest. Interesting
phenotypes did occur when the gene was
markedly overexpressed. In addition, how-
ever, genes would become expressed in
the wrong tissue or at the wrong place.
This ectopic expression was also a powerful
way to obtain phenotypic information. Ini-
tially, researchers were often confronted
with a high failure rate: gene constructs
would be integrated in high-copy head-
to-tail arrays (see Sect. 2), but would not
show a corresponding increase in expres-
sion level, or transgene expression would
diminish in consecutive generations. A
much-improved understanding of eukary-
otic gene regulation in the mid-1980s
provided the insight, leading to improved
construct design. For instance, DNA ele-
ments located tens of kilobases outside of
the β-globin gene were shown to control
high-level tissue-specific gene expression
through interaction with DNA sequences
immediately 5
-prime of the gene. Also,
sequence elements were discovered that
isolate a specific gene from influences
of flanking DNA. In addition, evidence
was obtained for a role of splicing in
mRNA stability. These advances led to the
use of much larger constructs harboring
the complete intron–exon structure of the
genes, and including large up- and down-
stream DNA segments. Such fragments
were obtained through cloning in cosmids,
bacterial artificial chromosomes and yeast
artificial chromosomes, allowing handling
DNA fragments in the range from 40 up
to 1000 kb.
1.2.2 Brief Overview of Developments
in Gene Targeting
The development of gene targeting by
homologous recombination in embryonic
stem cells (ES) can be attributed to two cru-
cial technological advances that ultimately
compounded. One line of research in-
volves ES research. Initially, ES cells were
used to study cellular differentiation and
cancer, because when injected subcuta-
neously into nude mice, teratomas would
develop. Figure 1(b) itemizes a number
of crucial advances in ES cell technology
that helped develop ES cell technology
to its current status. Improved culturing
conditions ensured sustenance of pluripo-
tence, a property of ES cells crucial for
their application as vehicles to introduce
germ-line mutations. Soon after the first
mouse was made that showed germ-line
transmission of genes introduced through
genetic modification of ES cells in vitro, evi-
dence was obtained that an endogenous
locus could be exchanged for a mutated
sequence by homologous recombination
in vitro (see Sect. 3). These strategies tar-
geted the murine HPRT gene and were
based on the fact that HPRT is an ex-
cellent selectable marker expressed in
the genome on the X chromosome of
male cells. Spurred by success, the ques-
tion arose whether gene targeting would
also be feasible for nonselectable auto-
somal genes; this was proven possible
shortly after.

Sections 2 and 3 will elaborate on
the conventional transgenesis and gene-
targeting technology. Noteworthy, with the
introduction of novel concepts in molec-
ular genetics, our ability to manipulate
gene expression in the mouse currently
knows few limitations. Sections 4, 5, and
7 of this chapter will elaborate on some
of the more recent technological advances
such as binary systems (see Sect. 4), ran-
dom mutagenesis (see Sect. 5), and RNA
interference (see Sect. 7).
2 The common denominator of these stud-
Transgenesis; Basics and Technology
2.1
Transgene Design
Shortly after its introduction in the early
1980s, the main scientific applications
of transgenesis in the mouse could be
roughly divided in two areas: (1) studies
of gene promoter activity (and/or other
regulatory elements) and (2) studies of
the effect(s) of gene overexpression (or
a mutated form thereof; Fig. 2a). Gene
activity is, simply put, controlled by a
promoter. A promoter is a stretch of
DNA sequence, which usually precedes the
coding part of a gene. A promoters’ task is
to regulate gene expression: it controls the
‘‘when (at which time point) and where’’
(in what cells, tissues) of gene activity.
Eukaryotic genes also carry a termination
element. In order to function properly
in a eukaryotic genome, a transgene
minimally meets these requirements: it
carries a promoter, a coding part, and a
stop signal. The easiest way to read out
promoter activity in cell or a developing
animal is by placing a so-called reporter
gene downstream of it. Reporter genes
encode proteins, often found in lower
Transgenic Mice in Biomedical Research 513
organisms such as yeast, fireflies, or
jellyfish, which reveal their presence by a
microscopically or macroscopically clearly
visible mark (Fig. 2a, b). This approach is
standardly used to examine spatio-temporal
gene expression patterns and/or tissue
specificity of native promoter sequences
in vivo (Fig. 2b).
By far, transgenesis is used to achieve
and/or study the effects of overexpres-
sion (higher gene activity than normal)
or ectopic expression (gene activity in dif-
ferent cell types than normal) of a gene
of interest, or of a mutated form thereof.
ies is the analysis of the resulting altered
phenotype. This can be abnormal devel-
opment, altered physiology or behavior,
development of disease, and so on. The
choice of regulatory sequences (see be-
low) determines whether the expression
of a transgene follows the expression pat-
tern of its endogenous counterpart or is
limited to distinct cell types or particular
developmental stages.
2.1.1 Origin and Structure
of the Transgene
The origin of a transgene, or elements
therein, may range from prokaryotes or
from lower (e.g. reporter genes) to higher
eukaryotes, like mice or men. In ex-
perimental biomedical research, human
transgenes offer several advantages. Im-
portantly, many known genetic disorders
in humans have been extensively char-
acterized at the molecular genetic level:
mutations or deletions are charted and
such ‘‘diseased alleles’’ are readily avail-
able. In addition, most genes and proteins
are well conserved between mouse and
man. This, of course, is important when
the biological activity of a gene product de-
pends on proper interaction with other
cellular proteins. Finally, the sequence
514 Transgenic Mice in Biomedical Research
divergence between mouse (transgene re-
cipient) and human (transgene donor) of
a given gene can be used to determine
whether a mouse is transgenic or not, what
its copy number is (number of transgene
integrations), and whether a transgene is
actively expressed, since it allows for dis-
crimination between transgene and the
endogenous gene. Figure 3 gives an exam-
ple of such an analysis.
Most eukaryotic genes display a typi-
cal intron/exon structure, with only exon
sequences represented in mRNA. Trans-
genes, in general, are more reliably ex-
pressed if the intron/exon boundaries are
Construct type
1. Promoter X Reporter gene
2. Promoter Gene
(a)
(b)
(d)
preserved in a transgene construct. Solely
cDNA-based expression vectors (i.e. exons
sequences only) frequently show low ex-
pression levels and are often silenced.
Since many integrating DNA or RNA
viruses do not carry the eukaryotic in-
tron/exon structures, an expressed genetic
sequence without such boundaries may
be recognized as foreign and potentially
harmful by the eukaryotic host cell and
will be inactivated (silenced) by cellular de-
fense mechanisms. The native intron/exon
structure of a gene need not be preserved
in its entirety: indeed, inclusion of only
one intron in a transgene has been shown
Promoter studies
X Gene studies
(c)
(e)
 Transgenic Mice in Biomedical Research 515

to augment transgene expression. Many Matrix attachment regions (MARs), scaf-

successful transgene constructs include
both genomic and cDNA sequences de-
rived form the same gene (Fig. 4a).
2.1.2 Regulatory Elements
Any transgene comprises a number of
essential elements that control gene ex-
pression (Fig. 4b). The most basic and
essential elements are the promoter and
termination signals for transcription (RNA
synthesis). A promoter controls gene ex-
pression by providing binding sites for
proteins (RNA polymerase transcriptional
machinery, cell type–specific transcription
factors) that regulate gene transcription
(i.e. activation). The real-life situation is
often much more complicated than this;
many more regulatory elements exist that
all play a role in establishing stable and
faithful gene expression patterns. Among
these are enhancers, which typically act
in an orientation-independent manner,
fold attachment regions (SARs), Locus
control regions (LCR), chromosomal insu-
lators, and antirepressor elements. Some
of these elements are involved in sub-
nuclear localization of genes to areas of
active transcription and/or insulate gene
expression from influences of surround-
ing chromatin. Some of these autologous
(i.e. endogenous) regulatory elements may
actually be many thousands of base pairs
removed from the coding sequences of a
gene. Indeed, if the purpose of a study is
to examine faithful gene expression pat-
terns, for instance throughout embryonic
development, a transgene should include
such endogenous elements. The exact lo-
cation of such elements within a locus
is often not known and hence there is an
obvious advantage in using large DNA seg-
ments as transgenes. The development of
molecular vectors, which can harbor large
pieces of DNA, such as so-called artificial

Fig. 2 Transgenesis; concepts. (a) Original scientific applications of transgenesis in the mouse were
roughly divided in two areas: (1) studies of gene promoter activity (and/or other regulatory elements)
and (2) studies of the effect(s) of overexpression (or a mutated form) of gene X. Promoter studies
typically make use of reporter genes (green box) that reveal their presence through specific catalytic
activity. Often-used reporter genes include the green fluorescent protein jelly fish (b, d), bacterial
beta-galactosidase (c), (also see Fig. 3), and fire fly luciferase (d). (b) Transgene expression is
controlled by a keratinocyte-specific promoter (source image: http://www.mshri.on.ca/nagy/
cre.htm; Maatman, R., Gertsenstein, M., de Meijer, E., Nagy, A. et al. (2003) Aggregation of embryos
and embryonic stem cells, Methods Mol. Biol. 209, 201–230). (c) Differential expression of
beta-galactosidase (upper left to lower right): Lymphatic vessels (intestinal surface), marginal zones
between red and white pulp (spleen), bronchial epithelium (lungs), Large skeletal muscles (limb),
granule cell layer (cerebellum), Intervertebral discs (source images: Valenzuela, D.M., Murphy, A.J.,
Frendewey, D., Gale, N.W. et al. (2003) High-throughput engineering of the mouse genome coupled
with high-resolution expression analysis, Nat. Biotechnol. 21, 652–659). (d) Fluorescent images of an
X-linked enhanced green fluorescent protein (EGFP) transgene; transgenic male offspring (left) of a
mating between an X-linked EGFP transgenic female and a nontransgenic male have ubiquitous
green fluorescence in the skin owing to the presence of a single active X chromosome. Transgenic
female pups (right) have mosaic (tortoiseshell) green fluorescence in the skin owing to random
inactivation of one X chromosome (source image: Hadjantonakis, A.K., Dickinson, M.E., Fraser, S.E.,
Papaioannou, V.E. (2003) Technicolour transgenics: imaging tools for functional genomics in the
mouse, Nat. Rev. Genet. 4, 613–625). (e) An external image of adenovirus-encoded GFP gene
expression acquired from a nude mouse in the light box 72 h after portal vein injection (see color
plate p. xxviii).
516 Transgenic Mice in Biomedical Research

Transgene Promoter Transgene Viral LTR

← Probes
(a) ns ts

Copy number 10 5 1 0 - - 6 - 1 - 60 - 16

Transgene

(b) Probe → Transgene specific (ts)

Expression
wt 1 2 3 4 5 wt 1 2 3 4 5

28S

Transgene
Endogenous

18S

(c) Probes → Nonspecific (ns) Transgene specific (ts)

Fig. 3 Transgene construction and detection. (a) Schematic representation of a hypothetical

transgene. The transgene is derived from mouse genomic sequences (brown). Transgene
expression is under control of a general promoter and terminates in a retroviral long terminal
repeat (LTR) element. Probes used for detection of transgene DNA or RNA are depicted as a
brown box (nonspecific (ns) probe, which detects both the endogenous gene and the
transgene) or a blue box (a transgene-specific (ts) probe, which only detects the transgene).
(b) Copy number determination by Southern blot analysis with a ts probe. Bold print above
image represent DNA samples with known copy numbers, resp. 10, 5, 1, and no transgene
copies inserted; the remaining lanes show four founders with varying copy numbers (image
adapted from:(http://www.healthsystem.virginia.edu/internet/transgenic-mouse/
southerndesign.cfm)) (c) Detection of transgene expression by Northern blot hybridization
clearly demonstrates the need for transgene specific probes (right panel). Left panel: expression
signal of endogenous gene masks transgene expression (see color plate p. xxix).

chromosomes that can be propagated in heterologous. For instance, when overex-

yeast (YACs), phages P1 (bacterial viruses;
PACs), or bacteria (BACs) has been im-
perative for the cloning and expression
of such transgenic constructs. Exceedingly
large constructs (>500 000 bp) are first
transferred into embryonic stem cells (see
Sect. 3), which are then used to generate
transgenic mice.
Regulatory elements need not necessar-
ily be autologous in nature, they may be
pression or ectopic expression is required,
general type heterologous promoters (e.g.
viral promoters) and/or enhancers are
widely used. The use of heterologous
and autologous regulatory elements is
often combined. The first published trans-
genic mouse model displaying a specific
phenotype made use of the general-type
metallothionein promoter (pMT). The MT
promoter is inducible in vitro and in
 Transgenic Mice in Biomedical Research 517

Origin Control
Construct structure
DNA elements

1. prom cDNA p(A) Copy DNA Regulation

of choice

Genomic Endogenous
2. prom; 5′ UTR ex ex ex 3′ UTR; p(A)
DNA regulation

“Hybrid” Regulation
3. prom ex cDNA ex 3′ UTR; p(A) constructs of choice
(a)

Regulatory sequences

1. Control elements: Autologous

Heterologous

2. Expression profile: Ubiquitous

Tissue restricted

3. Expression control: Constitutive (high/low)

(b) Inducible (on/off)

Fig. 4 Transgene structure and regulation. (a) Schematic overview of basic

transgenic construct design. For all constructs (1,2,3), eukaryotic coding sequence
is the starting material. Regulatory sequences are cloned separately into the
construct, which is entirely cDNA based (1). Endogenous regulatory sequences
may be included when genomic DNA is used (2). A transgene can be tailored to
specific requirements. The hybrid genomic/cDNA construct depicted
(3) combines intro-exon boundary inclusion with facile cloning options: the use of
cDNA may reduce the size of a vector considerably. prom: promoter sequences,
ex: exon, cDNA: copy DNA, 3
UTR: 3-prime untranslated region, p(A):
polyadenylation signal. (b) Regulatory sequences in transgene design. Depending
on the specific purpose of the model, many different systems are available to
control transgene expression. As indicated, regulatory element can be autologous
(i.e. derived from an endogenous locus) or heterologous in nature (1). The
required transgene expression profile may need to be ubiquitous, or cell type
specific. This determines the choice of promoter (2). Finally, the model may
require controlled expression; instead of constitutive expression, inducible
expression would be preferred (3). (Figures adapted from: Voncken, J.W. (2003b)
Transgene design, Methods Mol. Biol. 209, 51–67).

vivo with glucocorticoids, heavy metals, or
bacterial endotoxin (LPS). Heterologous
promoters and other regulatory elements
are applied widely, and as indicated be-
fore, their incorporation into constructs
is determined primarily by the aim of
the animal model (see Fig. 2). Not only
general-type promoters, like the ones driv-
ing housekeeping genes (e.g. phosphoglyc-
erate kinase PGK) or other genes that are
widely expressed throughout eukaryotic
cell types (e.g. histones, ß-actin), but also
518 Transgenic Mice in Biomedical Research
viral promoters are often used to ensure
high and ubiquitous transgene expression.
LCRs are included in transgenic constructs
to both enhance transgene expression
and to achieve position-independent and
copy number–dependent expression of a
transgene, often with cell lineage–specific
enhancer activity. Finally, promoter choice
is often dictated by whether expression of
a transgene is deleterious to the mouse or
not. For example, many oncogenes play an
important role in embryonic development,
interfering with their normal expression
(pattern) results in embryonic lethality. To
bypass this problem, either tissue-specific
or inducible promoters (or combinations
of such regulatory systems are used (see
Sect. 4)).
Taken together, the origin of DNA, struc-
ture and choice of regulatory elements
for a transgene are largely determined
by the scientific aim of the experimental
model itself. For biomedical studies em-
ploying the laboratory mouse as a model
system, the use of human transgenes is
often preferred.
2.2
Pronuclear Injection
Transgenes can be introduced into living
cells in many different ways. Among the
most standard procedures in vitro (‘‘in the
test tube’’) are transfection methods either
using calcium phosphate/DNA precipi-
tates or liposomes (small DNA-containing
vesicles surrounded by a lipid membrane),
which are taken up by cells, electropo-
ration (electroshock), viral vectors (e.g.
adenovirus, retrovirus, which carry the
transgene), particle bombardment, and
microinjection. Some of these methods,
however, cannot be used in combination
with mouse oocytes. The very first proof-of-
principle report on germ-line transmission
of a foreign DNA sequence in the mouse
was achieved through retroviral transduc-
tion. As indicated above, viruses of this
class are often inactivated in eukaryotic
cells. Although recent technological de-
velopments in the field of transgenesis
again make use of retroviral constructs (see
Sect. 7), pronuclear injection has been the
golden standard for generating transgenic
animals over the last two decades.
The principle of pronuclear injection
is simple: a sterile DNA solution is pre-
pared, which contains a very low con-
centration of the transgene (nanograms
(10e-12 kg)/microliter (10e-15 l)). A very
fine glass needle containing the transgene
DNA solution is inserted into one of the
pronuclei and DNA solution is expelled
into the nucleoplasma (Fig. 5a, 6a). The
amount of DNA injected is extremely low
(femtograms (10e-18 kg)/picoliters (10e-
12 l)). Injected transgene DNA in the nu-
cleus tends to integrate at random into the
genome. Often, this occurs in head-to-tail
tandem arrays called concatamers. (Fig. 5b).
The repetitive nature of such concatamers
may lead to transgene inactivation, as does
the random character of the insertion: it
often causes transgenes to land in inactive
heterochromatin, which constitutes most
of a eukaryote cells’ DNA. The use of in-
sulator elements (see Sect. 2.1) or targeted
transgenesis (see Sect. 4) provides solu-
tions to these problems.
2.3
Biotechnological Procedures
Figure 6 gives an overview of the biotech-
nological procedures involved in the pro-
duction of transgenic mice. Because gen-
erating transgenic mice by pronuclear
injection is a rather inefficient process, a
relatively large number of fertilized oocytes
 Transgenic Mice in Biomedical Research 519

(a)

Transgenesis Transgene
concatamer

Chromatin Single random

transgene
(b) integration

Gene targeting
Homologous
recombination
Targeting
vector

(c) Endogenous locus

Fig. 5 Microinjection. (a) Pronuclear injection of DNA into a fertilized

one-cell-stage embryo. In the early stages following fertilization, two pronuclei
are visible in the zygote: one from the oocyte and one from the sperm cell;
these will eventually fuse to form a diploid nucleus. Both prefusion pronuclei
are clearly visible in the cytoplasm (the male pronucleus is mostly used for
injection, since it is the largest) (left panel; source image: Hogan, B.,
Beddington, R., Costantini, F., Lacey, E. (1994) Manipulating the Mouse
Embryo. A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York). Microinjection of genetically modified
ES cells into blastocysts; arrow indicates collapsed, injected blastocysts; ES
cells are clearly visible in the blastocoel (right panel). (b) Schematic
representation of random integration of transgenesis in the mouse genome:
transgene either integrated single or as concatameric head-to-tail arrays.
(c) Gene targeting is homology-driven: a targeting vector will exchange with an
endogenous locus by homologous recombination.
520 Transgenic Mice in Biomedical Research

Holding pipet Male Injection needle

pronucleus

Transgene
DNA

Medium droplet
(a) Slide Zona pellucida

Nucleus

Lentivirus +
transgene

(b)

Blastocyst

Mutant
ES cells

(c)

Fig. 6 Schematic representation of a number of techniques to generate

genetically modified mouse models. (a) Pronuclear injection of transgene
DNA. (b) Injection of infectious viral particle in the perivitellin space; the
advantages of using lentiviral shuttle vector are significant: it obviates
potentially harmful procedures otherwise used to introduce transgenes into a
cell (pronuclear microinjection, electroporation) and offers some degree of
control over transgene copy numbers. Lentiviral vectors are also used to
generate transgenic lines through infection of ES cells. One of the few
restrictions on recombinant lentiviral vector use is the size limitation on the
transgene inserts. (c) Microinjection of pluripotent, genetically modified ES
cells into blastocysts.

needs to be injected to obtain the so- transgene in their genome. A typical

called founder mice: transgenic founders transgenic study uses at least three ex-
developed in utero from successfully in- pressing transgenic founders to establish
jected zygotes that carry an integrated independent transgenic mouse lines from.

Convenient numbers of fertilized eggs
are obtained by superovulation, much the
same as way as in humans for in vitro fer-
tilization purposes: exogenous hormones
(follicle-stimulating hormone and luteiniz-
ing hormone analogs) are injected at a
48-h interval to achieve multiple ovulations
per donor female, which are subsequently
mated to fertile males. Simultaneously,
recipient females, the so-called foster fe-
males, are endocrinologically primed for
pregnancy by copulation to vasectomized
males (see Fig. 7). Microsurgery is carried
out under aseptic conditions and general
anesthesia. A number of different mouse
strains are useful for biotechnological pro-
cedures, many of which have their own
advantages and specific traits (see Sect. 1).
Detailed descriptions of the technology,
useful strains, and husbandry are to be
found in a number of media.
3
Gene Targeting; Basics and Technology
First established in the late 1980s and early
1990s of the last century, gene targeting is
now a well-established technology for gen-
erating genetically modified mouse mod-
els. Gene targeting allows for germ-line
manipulation at predetermined genomic
loci by a process called homologous recom-
bination: part of an endogenous allele is
replaced or mutated by vector sequences
that carry flanking sequences homologous
to the endogenous gene (Sect. 3.1). It is
in this aspect where gene targeting funda-
mentally differs from conventional trans-
genic technology: the possibility to change
existing genes offers a very important and
powerful extension of the molecular ge-
netic tools to create experimental animal
models in biomedical research. Whereas
Transgenic Mice in Biomedical Research 521
transgenesis asks for genes and their phe-
notypes to be dominant in nature (e.g.
expression of (mutated) oncogenes), gene
targeting now permits study of reces-
sive gene function (e.g. tumor suppressor
genes). The concept of gene targeting is
simple: eradication of a gene may reveal its
function by a resultant phenotype. Current
applications range from conventional null
mutation of a given gene, the so-called gene
knockouts, to replacement of endogenous
alleles with mutated alleles and even in-
ducible (conditional; see Sect. 4.3) genetic
modifications.
3.1
Targeting Vector Design
Generally speaking, two types of vectors
exist by which genes can be mutated.
1. Replacement vectors
2. Random insertion vectors
All classical replacement type vectors have
in common that they carry stretches of
DNA, which are highly homologous or
identical to the gene, which is to be tar-
geted. The mechanism by which gene
replacement is accomplished, homologous
recombination, uses these identical or very
similar sequences of DNA: homologous
recombination is a sequence homology-
initiated and driven process (Fig. 5c, 8a).
It appears to be a highly selective pro-
cess, which only takes place between
relatively large (several kilobase pairs)
DNA segments. In fact, it is important
that the constructs used for homologous
recombination are being derived from
the exact same genetic background (iso-
genic) as the cell line used for gene
targeting. This requirement implies that
a sequence divergence of around 1 in
100 bp will cause a marked drop in
522 Transgenic Mice in Biomedical Research

Zygote donor

(a)
Microinjection
Foster

X
Oviduct transfer

(b) Vasectomized

(c) Transgenic and nontransgenic progeny

Fig. 7 Biotechnological procedures in conventional transgenesis. (a) Hybrid

F1 animals are used for superovulation and mating to generate sufficient
zygotes for injection. (b) Upon microinjection, the embryos are transferred
into the oviduct of a pseudopregnant female; pseudo-pregnancy, is achieved
by mating to a vasectomized male and exists for a maximum of 3 days; the
surgical transfer of microinjected one- or two-cell-stage embryos directly into
the fallopian tube at 0.5 day post coitum ensures sustenance of the pregnant
state. (c) Offspring will manifest coat colors of both strains (e.g. C57Bl6 and
CBA) used to generate the F1 hybrids (e.g. B6CBA/F1). Roughly, 10% of the
offspring will carry an integrated transgene, and will be further examined for
transgene expression and transmission. Such an animal is referred to as a
transgenic founder. In addition, these media provide comprehensive
information on historical and genetic backgrounds of in- and outbred strains
on mouse embryology and dissection of specific developmental stages.

the frequency of homologous recombi-
nation. Conventional gene-targeting con-
structs mostly harbor a selection marker
(neomycin, hygromycin, or puromycin re-
sistance gene), which serves a number
of purposes: it replaces endogenous gene
Functional domain
Protein
R R
Probe
R R
Select
(a) Interruption & selection
Genotype: +/+
(R-R fragments)
(b)
Fig. 8 Basic design of a conventional targeting
vector. (a) The targeting vector duplicates part of
the locus to be mutated (homologous
sequences), but is interrupted by a positive
selection marker (select), for instance, an
antibiotic resistance gene. Many conventional
targeting vectors also contain a negative
selection marker. The herpes simplex virus
Thymidine Kinase gene is often included: the TK
gene product converts a harmless compound,
for instance, Ganciclovir, into a cytotoxic
substance. The positioning of the TK cassette at
one of the ends of a targeting vector results in
exclusion of TK from the genome upon
homologous recombination, whereas TK is
cointegrated in case of random integration of a
targeting vector. (b) The vector is designed such
Transgenic Mice in Biomedical Research 523
sequences, thereby interrupting the gene
and rendering it nonfunctional (Fig. 8a).
In addition, the marker is used to positively
select for embryonal stem cells that have
integrated the targeting vector. A second
feature often included in a replacement
Gene product
Locus
Homologous
recombination
Targeting vector
−/− +/−
Wild-type allele
Null allele
that by means of an ‘‘external probe’’ (blue-grey
box: probe; from within the to be targeted locus,
but outside the homology regions used in the
targeting construct) and strategically chosen
restriction endonuclease sites (R) a shift is
detected upon homologous recombination. The
figure shows the three possible genotypes; two
wild-type (+/+) alleles, two knockout (−/−)
alleles (homozygous knockout), and a
combination of both in the heterozygous (+/−)
condition (see also Fig. 10). Mutagenesis vectors
may be introduced into ES cells via several
means; the most commonly used method is
electroporation (electroshock) by which the
cellular membrane is damaged, allowing uptake
of DNA in cells (see color plate p. xxvii).
524 Transgenic Mice in Biomedical Research
vector is a negative selection marker, for ex-
ample, the Herpes simplex virus thymidine
kinase (HSV-TK). Upon positive (negative)
selection, a number of separately isolated
ES cell clones are ‘‘genotyped’’ to ensure
proper recombination on both ends of the
targeting vector. Typically, these cells carry
a monoallelic mutation, that is, only one
of two alleles in the ES clone is replaced by
the targeting vector (Fig. 8b).
Random insertion type vectors insert at
will in the genome and, therefore, do
not rely on sequence homology with
target genes (see Sect. 5.1). Integration
of a vector in a locus may, however,
cause inactivation of a gene. The best-
known application of insertion type vectors
is gene trapping (see Sect. 4 and 5).
Gene trap vectors are constructed such
that they either trap promoter activity
or trap a polyadenylation signal from
an endogenous locus (Fig. 15). In this
manner, not only actively transcribed
but also inactive genes can be trapped
(with promoter-trap or poly(A)-trap vector
respectively). Variations on this concept
are described in Sect. 5.
3.2
Embryonic Stem (ES) Cells
In contrast to classical transgenesis, where
mutagenesis is done directly in the em-
bryo, gene targeting is done in embryonic
stem cells (ES cells) (Fig. 9a). These are very
early stem cells isolated from preimplan-
tation embryos (blastocysts). ES cells are
pluripotent, that is, they are fully undiffer-
entiated and, in essence, have the capacity
to participate in mouse embryogenesis and
become any cell type in the animal. The
first steps in a gene targeting experiment
are carried out in vitro. ES cell culturing
conditions are aimed at maintaining their
pluripotency. ES cells are either cultured
on a layer of supporting feeder cells (mouse
embryo fibroblasts (MEFs)) in the presence
of factors that block differentiation of stem
cells in general (like Leukemia Inhibitory
Factor,) or are ‘‘feeder-independent.’’ It
is their pluripotency that is ultimately
called upon: when generating targeted mu-
tations in the mouse, male mutant ES
cells are reintroduced into preimplanta-
tion embryos and made to participate in
embryogenesis and spermatogenesis in
particular. If the latter occurs, the tar-
geted mutation can be stably transmitted
via the germline to offspring and bred to
homozygocity.
3.3
Genesis of Mosaic Embryos
ES cells may be used in a number of ways to
generate genetically mosaic embryos. The
most commonly used procedure is blasto-
cyst injection: preimplantation blastocysts
are used to introduce ES cell into (Fig. 5a).
The donor mouse line (i.e. of which ES
cells are derived) and the recipient line (i.e.
of which blastocysts are procured) differ in
coat color genetics. Typically, an inbred
strain such as C57blk (black coat color)
is used for blastocyst production to in-
ject 129Sv or a 129Ola (sand-colored coat),
some of the most commonly used ES cell
lines, into. The procedure described above
is schematically represented in Fig. 6c.
Alternatively, ES cells can be fused to
morula stage embryos in a procedure
termed morula aggregation. Embryos in the
morula stage typically comprise 8 to 16
blastomeres (individual embryonic cells)
surrounded by a protective layer called
the zona pellucida. In order to achieve
aggregation, this zone is first removed and
the embryos together with a small number
of ES cells are cocultured in a small
depression, in which gravity forces them to

(a)
(c)
Fig. 9 Embryonic stem cells, embryos, and
chimeric mice. (a) Left panel: ES cells grown on
mouse embryonic fibroblasts (MEFs); white
arrows point at colonies of tightly coherent ES
cells; right panel: feeder-free ES cells.
(b) Aggregation of 8-cell-stage morulas (black
arrow) with genetically modified ES cells (white
arrow). (c) Chimeric mice beautifully displaying
the coat color mosaicism resulting from
contribution of ES cells of two different genetic
backgrounds to the developing embryo the mix
of, in this case, C57Bl (recipient
blastocyst – black) and 129Ola (donor ES
be in close contact with each other (Fig. 9b;
http://www.mshri.on.ca/nagy/cre.htm).
Both methods yield the typical mosaic
coat (e.g. a mixture of different coat colors,
like black and sand). The mice that display
Transgenic Mice in Biomedical Research 525
(b)
(d)
cells – sand) becomes manifest as Agouti,
patches of purely 129Ola-derived tissue is sand
colored (e.g. over left eye). (d) Electrofusion of
two-cell-stage embryos (middle left) results in
fusion and formation of one-cell-stage tetraploid
embryos (white arrows). Tetraploidy does not
hamper placenta formation, but does not permit
normal embryogenesis. Tetraploid blastocysts
injected with diploid ES cells will support fully ES
cell–derived embryonic development (source
image: (http://www.mshri.on.ca/nagy/cre.htm))
(see color plate p. xxxi).
the coat color mosaicism are referred to as
chimeric mice (Fig. 9c). Chimeric males are
bred to wild-type mice to generate a mouse
that is heterozygous for the mutant allele in
all its cells; the subsequent mating of two
526 Transgenic Mice in Biomedical Research

heterozygous animals allows the mutation
to be bred into a homozygous state.
3.4
Biotechnological Procedures
In contrast to transgenic technology,
preimplantation embryos are isolated at
the blastocyst stage. Microinjected em-
bryos are transferred directly into the
uterus of a 2.5-day pseudopregnant female,
where they develop into chimeric embryos
(Fig. 9b, 10). A typical targeting experi-
ment uses at least two independent mutant
embryonic stem cell lines for microinjec-
tion to verify and confirm the phenotype

Blastocyst donor

ES cell donor

Mutant
ES cells
(a)

Foster

(b)

X
Uterine transfer
Microinjection
Vasectomized

(c) Chimeric male

(d) Mutant and wild-type progeny

 Transgenic Mice in Biomedical Research 527

in two independently established homozy- been developed over the last decade are
gous knockout animals. described below.

4 Fully ES Cell–derived Embryos
Refinements to the Models
Transgenes or gene knockouts may cause
unexpected embryonic or neonatal lethal-
ity, either by interfering with embryo
development or placentation. If the aim
of an animal model is to, for example,
study the effect of a transgene or a gene
knockout on adult physiology or devel-
opment of a disease, embryonic lethality
limits the usefulness of such a model. To
circumvent this problem, animal molecu-
lar geneticists have sought to refine animal
models. The resulting mouse models of-
ten combine aspects of transgenesis and
gene targeting and may include a molec-
ular switch, by which null mutation of a
gene now is rendered conditional. Such
on/off models are referred to as binary
models. The most pivotal advantages of
such molecular switches are gain in ac-
curacy and specificity of the model and
an important concomitant reduction of
disease burden for the animals. Some ex-
citing models and applications that have
4.1
The placenta is one of the first organ
systems to develop during early embryo-
genesis. It provides an essential means
by which the developing embryo and the
mother animal exchange essential factors
such as nutrients and oxygen. A surpris-
ingly large number of null mutations result
in defective placentation. Defective placen-
tation in a conventional genetic approach
thwarts all possibilities to uncover a gene
function during early embryogenesis. J.
Rossant and coworkers developed an ele-
gant solution to this problem during the
early 1990s: tetraploid embryos were used
to reintroduce diploid ES cells into. The
concept is elegantly simple: tetraploid cells
will contribute to placentation (polyploidy
is a normal feature within the developing
placenta); they will, however, not con-
tribute to the development of the embryo
itself. It was shown that in this approach,
reintroduced normal diploid wild-type ES
cells were fully capable of supporting nor-
mal embryonic development. The data
demonstrated that with this technology

Fig. 10 Biotechnological procedures in conventional gene targeting. (a) Inbred strains are used for
superovulation and mating to generate sufficient blastocysts for injection. The procedures in use to
obtain sufficient blastocysts for microinjection are natural matings or superovulation. Breeding
properties of some inbred strains are relatively poor, hence, natural mating often require large
numbers of mating pairs. Super ovulation is often not very efficient in inbred strains either, but will
usually yield adequate embryo numbers for injection. (b) Upon microinjection, the embryos are
transferred into the oviduct of a pseudopregnant female (see also legend to Fig. 7). (c) Since male ES
cells are used, researchers look for a gender bias among chimeric offspring: a potent male (XY) ES
cell line forces even female recipient blastocysts (XX) into a male sex phenotype. As a general rule is
considered: the higher the coat color mosaicism, the more likely it is that the ES cells participate in
spermatogenesis, the higher the chance for germ-line transmission of the mutation; (d)–Germ-line
transmission is revealed by the coat color of the donor (ES cell; sand-colored in the figure; several ES
cell lines are in use) strain; 50% of the sand-colored offspring will be heterozygous for the germ-line
mutation (see color plate p. xxx).
528 Transgenic Mice in Biomedical Research

the study of genetically manipulated ES to form one tetraploid one-cell-stage em-

cells in all fetal lineages was possible,
while, at the same time, potential negative
effects of a null mutation on placental de-
velopment were circumvented. Tetraploid
recipient embryos were generated by elec-
trofusion: normal diploid two-cell-stage
embryos were subjected to a defined elec-
troshock that results in fusion of the cells
bryo (Fig. 9d). This tetraploid early embryo
is allowed to further develop in vitro, to be
used at a later stage for aggregation or
microinjection (see Sect. 3.3). It should be
noted that although the role of a gene
can now be studied independent of pla-
centation, specific lethality as a result of
gene mutation is not prevented. To bypass

Transgene 1

Inactive
oncogene rtTA X

tetO Oncogene
IE-CMV

rtTA rtTA + Tetracycline

rtTA

rtTA
Transgene 2 rtTA

(a) Transactivator Promoter A rtTA

Transgene 1

Inactive
oncogene X

Promoter B STOP Oncogene

LoxP
Transgene 2

Recombinase Promoter A CRE

(b)

4OHT
CRE-MER
Hsp
(c) Cytoplasm Nucleus
 Transgenic Mice in Biomedical Research 529

these problems, inducible or binary mod-
els are used.
4.2
Conditional Transgenesis
Conditional transgenesis is employed to
have better control over (trans)gene ex-
pression in a given model. This may be
advantageous for a number of reasons, for
instance, to circumvent lethality during
embryonic development caused by toxic or
teratogenic properties of transgenes or to
study the onset of disease from its earliest
stages onward.
In its simplest form, conditionality is in-
tegrated in an animal model by means of
tissue-specific promoters and/or control
element usage (see Sect. 2.1). Transgene
expression is confined to a defined target
tissue or select cells within a tissue, for
instance, epithelial lung cells (surfactant
protein promoter) or anterior pituitary
cells (POMC, pro-opiomelanocortin pro-
moter). Several tens to hundreds of mod-
els have been created and used in this
fashion to successfully study the specific
effect(s) of transgene expression in devel-
opment or disease.
Truly conditional transgenesis, in which
the investigator not only determines where
a transgene is turned on but also when
and how strong, can be achieved with bi-
nary models. Binary models incorporate
a switching mechanism. Several molec-
ular switches exist to turn on or off
transgenes. Figure 11 lists examples of
these methods. Most binary switches
are reversible. Two main concepts are

Fig. 11 Binary models. (a) The Tet-system requires two independently generated transgenic lines,
which are combined by interbreeding. Transgene 1 carries a minimal (‘‘enhancer-less’’)
Cytomegalovirus immediate early (IE–CMV) promoter fused to heptamerized prokaryotic tetO
regulatory sequences. Transgene 1 is transcriptionally silent until the transactivator line (transgene 2)
provides transactivator protein. In the original Tet-system, transgene 2 expressed a tetracycline
(Tc)-controlled transcriptional activator, tTA. tTA is a fusion between the Tet-repressor of the Tn10 Tc
resistance operon of E. coli and a C-terminal part of the herpes simplex transactivator protein VP16.
tTA will induce transcription from the tetO-controlled transgene up to five orders of magnitude, but is
inhibited by Tc. The example depicts rtTA (red ovals), a reverse Tc-controlled transactivator, which is
a mutant form of tTA and needs Tc-derivatives to bind tetO (yellow squares). So rather than an
inhibitable model, rtTA is an inducible model. Refinements on the Tet-system include choice of
regulatory sequences (promoter A) in transgene 2 (cell type–specific, general promoter). In addition,
several derivatives of Tc exist, among which Doxocyclin (Dox) has better tissue penetration and
pharmacokinetic properties than Tc itself and very high and selective affinity for TetR. (b) The use of
bacteriophage P1 CRE-LoxP recombination–dependent removal of a strong transcriptional block
(STOP) in a transgene construct may be helpful in defining the molecular events at the onset of
tumorigenesis and in addition circumvents possible embryonic lethal effects of the transgene. The
choice of regulatory sequences (promoter A) in transgene 2 is determined by the model. Although the
excision of a transcriptional block is, in principle, reversible, for practical reasons this system is rarely
used as a binary switch, since it would entail obligatory screening for genetic modifications
(insertions) in target cells: this obviously compromises the usefulness of the models in terms of
precision and rapidity. (c) Fusion of CRE to the mutant forms of the ligand-binding domain of nuclear
hormone receptors, such as the estrogen receptor (MER), has generated a recombinase CRE-MER
that can be ‘‘induced’’ at the posttranslation level: CRE-MER is retained cytoplasmic through
interaction with heatshock proteins (Hsp) such as HSsp90; upon addition of 4-hydroxytamoxifen
(4OHT), a synthetic steroid, the Hsp-binding is released, and the recombinase shuttles to the
nucleus to excise floxed sequences (also see Fig. 12) (see color plate p. xxxii).
530 Transgenic Mice in Biomedical Research
used: (1) a combination of nonmammalian
promoters and transcription factors and
(2) integration of transcriptional interrup-
tions that are removed by recombination.
The use of inducible transcription by
means of nonmammalian, for example,
regulatory elements borrowed from bacte-
ria or lower eukaryotes, like the fruit fly (D.
melanogaster), allows transgenes to be ac-
tivated and silenced ‘‘on command.’’ The
system that utilizes removal of a strong
transcriptional block by means of recombi-
nation, although in principal reversible, is,
for practical reasons, mostly used one-way.
Several binary switches are item-
ized below:
– Tetracycline-controlled transgene expres-
sion: the Tet-system. The transgene of
interest is placed under the control of
a Tetracycline resistance operon from
Escherichia coli, the tet-operator (tetO).
A second, independent transgenic line
expressing a derivative of TetR, a tran-
scriptional regulator of tetO, confers
Tetracycline (Tc)-inducibility or repres-
siveness to the transgene of interest
(see Fig. 11a). Transcription factors
with regulatory activity toward the
tetO are absent in eukaryotic cells,
which means that theoretically the sys-
tem cannot be leaky for transcription
and pleiotropic effects are therefore
not expected.
– Insect hormone–controlled transgene ex-
pression. The system uses the molting
steroid hormone ecdysone for transgene
induction. It requires a heterodimeric
receptor and an ecdysone responsive
element, which is incorporated as a reg-
ulatory element in the transgene of in-
terest. Administration of ecdysone (or a
derivative muristerone A) rapidly and se-
lectively induces transgene expression.
Upon withdrawal of the steroid hor-
mone, expression is silenced. Again,
since mammalian cells lack insect hor-
mone receptors, the binary system
theoretically works as an on/off system,
much like the Tet-system. Ecdysone
nor its derivatives are toxic or terato-
genic. Owing to excellent pharmacoki-
netic properties, the method is ideally
suited for very precise conditional con-
trol over gene expression.
– Transgene induction by removal of a tran-
scriptional block. The system makes
use of a recombination process de-
rived from bacteriophage P1: phages
(bacterial viruses) use a site-specific
recombinase (CRE) to integrate their
DNA into the genome of their bacterial
host. An essential second component
of this process is a recombinase recog-
nition site, called an LoxP site: two LoxP
sites are recognized and recombined by
CRE (Fig. 12a). The two essential com-
ponents, LoxP and CRE, also work in
eukaryotic systems, and are commonly
maintained on two separate transgenic
lines. Flanking a sequence with two
similarly oriented LoxP sites will lead
to its excision in the presence of CRE.
Hence, an on/off transgenic system
employs a silenced transgene, in which
a strong transcriptional block is brack-
eted by two similarly oriented LoxP
sites; a second independent transgenic
line, which expresses the CRE recom-
binase, may be driven off a general,
tissue-specific or inducible promoter,
depending on the objective of the
study (Fig. 11b). More details on this
recombination-based switching mech-
anism will be discussed in Sect. 4.3.
4.3
Conditional Gene Targeting
Gene targeting is widely used to study
gene function in the mouse. Hundreds
 Transgenic Mice in Biomedical Research 531

CRE-LoxP recombination

Bacteriophage DNA

Bacterial DNA
LoxP + CRE Insertion

Excision + CRE

(a)
Wild-type allele

Conventional Null allele

select

Conditional Floxed allele

LoxP
Null allele

(b)

Fig. 12 (a) CRE-LoxP system: LoxP sites are short sequences harboring inverted 13 nucleotide
repeats separated by an 8-nucleotide asymmetric spacer. The LoxP are recognition sites for the
recombinase CRE. CRE drives site-specific recombination of two identical LoxP sites, for clarity
depicted as a yellow and a black triangle. Two similarly oriented LoxP sites on one contiguous
DNA strand will be recombined by CRE and the sequence in between the LoxP sites is excised.
(b) This recombination principle is used in conditional gene-targeting vectors: a crucial coding
region (two blue exons) is ‘‘floxed’’ (i.e. flanked by LoxP sites) in ES cells and excised in vivo. The
LoxP sites in the ‘‘floxed’’ allele are assumed not to interfere with wild-type gene expression. The
figure compares the structure of a conventional and a conditional knockout allele. A variation on
this theme is the FLP-frt system, which is derived from yeast. The FLP gene product, like CRE, is
a site-specific recombinase; frt (FLP recognition target) represents the small inverted repeats,
analogous to LoxP, recognized by the recombinase (see color plate p. xxxiv).
532 Transgenic Mice in Biomedical Research
of genes have been ‘‘knocked out’’ in
the conventional approach, in which the
null mutation is transmitted through the
germline, as discussed in Sect. 3.1. An
estimated one in three null mutations
results in embryonic or early postpartum
lethality. Therefore, germ-line mutations
may be appropriate models to recapitulate
human congenital genetic disorders. They
may, however, not be the best tools to
study gene function in adult mice, since
this is hampered by often unexpected and
unexplained lethality. K. Rajewsky and
coworkers were among the first to develop
a conditional gene-targeting strategy that
allows inactivation of a gene in a defined
spatio-temporal setting: the null mutation
can, for instance, be confined to selected
cell types or to a specific stage during
development.
Conditional gene targeting makes use
of the CRE-LoxP principle, as described
in Sect. 4.2. Briefly, LoxP sites flank a
genomic sequence in a targeting vector,
which will be deleted in vivo; this allele
is referred to as a floxed allele (Fig. 12b).
This final ‘‘floxed’’ wild-type allele only
carries recombinase recognition sites at
positions within the locus where they are
presumed inert, for example, introns: up
until the moment of recombination-driven
excision in vivo, the allele should function
as a wild-type allele. Since the CRE-LoxP
system is applied more widely than the
FLP-frt system in vivo, further descriptions
of conditional gene targeting will focus
mainly on the CRE-LoxP system.
The current standard experimental con-
ditional approach requires two separate
mouse lines:
1. the ‘‘floxed’’ line (as described above)
2. the ‘‘deleter’’ line, expressing CRE.
The transcriptional control directing
CRE expression determines the specificity
and degree of regulation of the condi-
tional knockout. CRE-transgene expres-
sion is controlled either by constitutively
cell type–specific or inducible regulatory
elements (also see Sect. 2.1 and 4.2).
Deleter strains are most often generated
by conventional transgenic technology. Al-
ternatively, the CRE-cDNA can be inserted
via targeted mutagenesis to a locus of in-
terest, under control of the endogenous
promoter. Although the latter method
does not require a thorough knowledge
of the regulatory elements controlling the
locus at hand, the approach is not as
straightforward as conventional transge-
nesis. Drawbacks of the former method
comprise all known problems linked to
conventional transgenesis: transgene ex-
pression is dependent on integration site
and copy number. Consequently, a num-
ber of independent founders will have to be
generated to select a useful strain. A sub-
stantial collection of tissue-specific CRE
mouse lines is currently available.
Inducible CRE expression allows for
a high level of control (see Sect. 4.2).
Again, it should be noted that conditional
gene targeting is mainly used unidirec-
tionally, that is, once a gene of interest is
inactivated through CRE-mediated recom-
bination, reversion of the genotype is not
easily accomplished.
4.3.1 Controlled CRE Expression
Inducibility can be conferred by rather
simple measures such as CRE expression
through infectious viral particles. Such
viruses can be locally administered or
systemically. In addition, their tropism (i.e.
host-cell range) can be manipulated.
Inducible mammalian promoters have
been used, as well as the Tet-system (see
Sect. 4.2). Besides the induction of CRE

transcription and translation, CRE induc-
tion has also been achieved at the post-
translational level. This was accomplished
by fusing CRE-encoding sequences in
frame to the ligand-binding domain (LBD)
of nuclear steroid hormone receptors, such
as the estrogen or progesterone recep-
tor (ER, PR respectively). Ligand binding
transfers the CRE-ER fusion product into
the nucleus, where the recombinase exerts
its catalytic activity toward LoxP sites. Mu-
tation of the ER moiety, has generated
CRE-MER (mutated estrogen receptors)
fusions, which are only induced by syn-
thetic hormones and not by their natural
ligands (Fig. 11c). Ongoing improvements
of these and alike inducible systems clearly
generate a highly sensitive and specific
means to control transgene expression and
enable the study of gene ablation and
overexpression that, without such tools,
were impossible. An increasing number
of published models employing CRE-MER
variants attest to the feasibility of the ap-
proach in vivo.
4.4
Knockins: Humanized Mice
Many studies in human disease have un-
covered mutations that are relevant to
the etiology of the pathological condition.
Examples of these are large congenital
chromosomal duplications or deletions
in inborn genetic disease, specific dom-
inant mutations in oncogenes, recessive
mutations in tumorsuppressor genes, or
polymorphisms in genes involved in lipid
metabolism linked to atherosclerosis. In
order to understand the exact molecular
mechanism underlying the development
of disease, such mutations need to be
exactly recreated in the mouse. Clearly,
conventional and conditional genomic ma-
nipulations are not specific enough to
Transgenic Mice in Biomedical Research 533
address such issues. CRE-mediated re-
combination has provided possibilities and
strategies to manipulate the genome be-
yond conditional gene knockouts or trans-
genesis. As illustrated in Fig. 13 and 14,
the CRE-LoxP system can be used to ma-
nipulate the genome in various manners:
– Gene knockin or gene exchange strategies
apply CRE-LoxP-mediated recombina-
tion to exchange endogenous coding
regions for exogenous or mutated se-
quences in a locus (Fig. 13a). Espe-
cially, the latter application is of signif-
icant interest to the scientific commu-
nity, since it permits the development
of animal models, which reflect hu-
man genetic disorders more closely
than models applying overexpression
or ablation of a gene. Initial ap-
proaches inserted reporter genes into
an endogenous locus; this simple vari-
ation on conventional gene targeting
achieves the same purpose as a trans-
gene harboring a reporter preceded
by endogenous regulatory sequences
(see Sect. 2.1). The main difference, of
course, is that this strategy obviates
the handling of large DNA fragments
with potentially unidentified regulatory
elements needed to achieve faithful ex-
pression patterns, since these elements
are provided by the targeting into the
endogenous locus. Recently developed
strategies take knockin significantly
further and allow for specific intro-
duction of (point) mutations and re-
peated exchange of genetic sequences:
recombination-mediated cassette ex-
change (RMCE) applies strategically
positioned pairs of wt and mutated
LoxP sites or LoxP and frt sites to
achieve stepwise and directional re-
placement of genomic sequences with
other of mutated sequences (Fig. 13b).
534 Transgenic Mice in Biomedical Research

Wild-type locus

Homologous
recombination

Select Targeting vector

= Point mutation LoxP

Knockin allele

(a)
Wild-type locus

(1.) Targeted allele

Select

Exchange
Select cassette

(2.) Targeted allele

Select

(3.) Modified allele

(b)

Fig. 13 Applications of site-directed recombination in molecular mouse genetics. (a) A

conditional targeting construct usually harbors an antibiotic resistance gene (select) flanked by
recombinase recognition sites (either LoxP or frt; see legend to Fig. 12). Prior to microinjection
into blastocysts or aggregation with morulas, the selection marker is excised in ES cells by
recombination (by transient expression of either CRE or FLP). The targeting construct depicted
contains a specific point mutation (asterix) in one of its exons. Homologous recombination
results in introduction of the mutation in the endogenous locus. (b) The use of multiple
nonidentical LoxP and or frt sites (black, red, and blue triangles) creates the possibility to
repeatedly exchange sequences between vectors and a locus via site-specific recombination.
The procedure is referred to as recombination-mediated cassette exchange (RCME). First, a locus
is targeted and an asymmetrically ‘‘floxed’’ selection marker is introduced (1). Using the same
LoxP variants, an exchange cassette is introduced (2) upon which the selection marker is
removed by recombinase-mediated excision (3). The exchange procedure can be repeated
multiple times, since the position of the most peripheral recombinase recognition sites (black
and red triangles) is maintained (see color plate p. xxxv).
 Transgenic Mice in Biomedical Research 535

LoxP LoxP

CRE CRE
LoxP LoxP

(a) Deletion Inversion

LoxP

LoxP
Lymphoid
CRE

(b) Translocation

LoxP

Testis/zygotene
LoxP CRE

(c) Monosomy Trisomy

Fig. 14 CRE-LoxP applications in chromosome engineering. (a) Chromosome engineering
is an important tool that facilitates functional analysis of large chromosomal regions: large
segments of DNA can be either deleted or inversed, depending on the orientation of the
LoxP sites in respect to each other. Series of overlapping deletions can be generated by
combined use of one targeted LoxP site and retrovirus-mediated random insertion of
additional LoxP sites. CRE-LoxP recombination may be used to generate balancer
chromosomes to maintain lethal recessive mutations and has the potential to facilitate
large-scale mutagenesis screens. Deletion or inversion carriers can be used to uncover and
instantly map, for example, ENU-induced recessive phenotypes to deleted or inversed
chromosomal regions. (b) Interchromosomal recombination by positioning LoxP sites on
nonhomologous chromosomes will recreate translocations, which occur in human disease.
(c) The use of CRE-mediated recombination to create defined autosomal trisomies in the
mouse, for example, through meiosis-/zygotene-specific CRE-deleter strains that are solely
active in testis.
536 Transgenic Mice in Biomedical Research

– Chromosomal deletions, inversions, trans- has been reported on in the scientific

locations. Simultaneous specific posi-
tioning of LoxP sites on one chro-
mosome allows for gene inversions
or complete deletions, depending
on the orientation of the recom-
binase recognition sites relative to
each other (Fig. 14). Positioning of
such recombinase recognition sites
on nonhomologous chromosomes has
proven to be a feasible strategy to
achieve chromosomal translocation,
alike translocations that are implicated
in some leukemias.
– Targeted integration of transgenes to loci,
which are known to be ‘‘open,’’ that
is, not embedded in heterochromatin.
The gain of this approach is a high
probability of transgene expression. An
often-used target locus is the ROSA26
locus, an ubiquitously transcribed lo-
cus that does not encode a protein,
but which is active in many different
cell types throughout development and
adult life. Transgenes are flanked by
DNA sequences homologous to the
ROSA26 locus and targeted via con-
ventional methods (see Sect. 3).
– A substantial number of other appli-
cations for site-directed recombination
literature. Since the principal mecha-
nism remains the same in all instances,
a few of these applications are listed
here, but not worked out in detail:
selection marker recycling, exchange of
reporter genes, removal of reporter genes,
or selection markers (as in the generation
of floxed alleles).
5
Random Mutagenesis in ES Cells
The completion of the mouse and human
genome sequences has sparked renewed
interest in developing tools for genome-
wide mutagenesis in the mouse. Among
the main molecular genetic technologies
currently pursued are targeted mutagen-
esis (see Sect. 3 and 4), insertional muta-
genesis, by insertion of gene trap vectors
or viral or transposable sequences, and
chemical mutagenesis. These technologies
all represent random mutagenesis proto-
cols, which, in principle, can be applied
to mouse ES cells. The important advan-
tage of random mutagenesis is that new
mutations can be generated in mice at

Fig. 15 Random mutagenesis strategies. (a) Promoter-trap and poly(A)-trap vectors, respectively,
catch or induce transcriptional activity and have the potential to identify most, if not all, of the active
and inactive genes in the genome, including alternatively spliced forms and low-abundance
transcripts, and is thus an important tool in genome annotation. (b) Gene mutation through random
insertion of retroviral vectors (or transposable elements; see Sect. 5.2). Shown is an oncogene, which
is activated by a nearby proviral insert, and a tumorsuppressor gene, which is inactivated proviral
insertion into the gene. Inserted elements are engineered such that they simultaneously function as
sequencing tags to identify the surrounding genomic DNA. (c) Stable or inducible RNA interference
as an alternative to targeted mutagenesis to study gene function in mice: a short double-strand RNA
(ds) stem-loop-stem structure (white arrows, blue loop) is produced from a RNA polymerase
III-promoted vector. The loop is enzymatically removed inside the cell; the resulting short interfering
RNAs will bind their complementary mRNA, upon which the target mRNA is degraded by a complex
called RISC (RNA-induced silencing complex). RNA interference–mediated gene silencing does not
require targeted mutagenesis of a gene, and may therefore represent a more efficient and rapid
method to assess gene function (see color plate p. xxxvi).
 Transgenic Mice in Biomedical Research 537

a rate far exceeding conventional gene 5.1

targeting. In addition, it is possible to
identify and maintain recessive lethal phe-
notypes at any developmental stage, which,
for instance, is not possible with chemical
mutagenesis (see Sect. 5.3).
Gene Trap Mutagenesis
Gene trapping provides a method to
mutate genes in the genome through ran-
dom insertion throughout the genome.

SA p(A)

Promoter Select
Promoter trap

Splice

SD p(A)

Promoter Promoter Select

poly(A) trap
(a) Splice

Activation
Provirus

Proto-oncogene

Tumor Suppressor gene

(b) Interruption

Pol III prom dsRNA

AAAAA mRNA

Degradation
(c) (RISC)
538 Transgenic Mice in Biomedical Research
Essentially, all currently designed gene
trap vectors insert a selectable marker
or reporter gene lacking essential regula-
tory sequences into an endogenous gene.
Through marker insertion the gene is
interrupted, while marker expression is
complemented by endogenous regulatory
elements (Fig. 15a). In this manner, tran-
scriptionally active as well as inactive loci
can be trapped. Recent reports provided
proof that gene trapping can also be used
to selectively mutagenize genes that share
certain properties, in this case, the fact that
their gene products are all transmembrane
proteins. A ‘‘secretory’’ trap vector was
constructed such that it captured signal
sequences in preceding exons by means of
including a ‘‘capturing’’ transmembrane
domain in the gene trap construct. These
and other approaches collectively demon-
strate the power and versatility of gene
trapping in genome-wide functional anal-
ysis of molecular mechanisms important
in development and physiology. A col-
lective gene trap database has recently
been initiated by a number of consor-
tia (http://www.igtc.ca/index.html – http:
//www.escells.ca/ – http://www.mmrrc.
org/ – http://tikus.gsf.de/project/web
new/index.html – http://www.sanger.ac.
uk/PostGenomics/genetrap/ – http:
//baygenomics.ucsf.edu/overview/
welcome.html).
5.2
Retroviruses and Transposable Elements
in Random Mutagenesis
The use of retroviruses in random muta-
genesis is adapted from applications in
which proviruses are used to screen for
collaborating oncogenes in leukemogene-
sis. Slow transforming retroviruses such
as Moloney Murine Leukemia Virus (Mo-
MuLV) cause transformation by integra-
tion into the genome. Proviral integration
may activate oncogenes or interrupt tu-
mor suppressor genes (Fig. 15b). This is
an extremely useful asset in random mu-
tagenesis, since the integrated proviruses
now serve as a molecular tag for cancer
genes: it can be used to identify the locus
at which it is integrated by sequencing the
flanking integration site. Retroviral vectors
have been adapted for use in mouse em-
bryos and ES cells as well, and now permits
germ-line mutagenesis.
In analogy to random proviral insertion,
also transposable elements from yeast, in-
sects, fish, and mammals, among other
organisms, can be used for functional ge-
nomic analyses. Transposable elements
(TEs) are a heterogeneous class of ge-
netic elements that have the capacity to
move from one site on a chromosome to
another: they ‘‘jump.’’ TEs are very effi-
cient at integrating into DNA, and some
elements can carry extra DNA. TEs are
therefore useful vectors for transferring
new genetic material into genomes and
for random insertional mutagenesis. By
way of example, among the properties that
make the Tc1/mariner superfamily of TEs
useful for molecular genetic research are
the facts that they are easy to work with,
their efficient and stable integration, and
persistent, long-term transgene expres-
sion following transposon-mediated gene
transfer. Importantly, such TEs ‘‘jump’’
in species other than their original hosts,
which underscores the need for fur-
ther development and use of these and
alike molecular tools for functional ge-
nomics in the mouse. Several reports on
germline induced TE transposition and
germline attest to the mutagenic potential
of the approach.

5.3
Random Enu Mutation
For many years, mutant mouse models
were obtained largely as a ‘‘spin-off’’ from
research on the mutagenic properties of
chemical compounds and radiation. With
the recent advent of more genome re-
sources, it has become possible to identify
such induced mutations more rapidly. Ini-
tially, substantial efforts were undertaken,
involving collaborations of many labora-
tories, each with their own phenotyping
expertise. At present, robust technology
is in place to mutagenize mice. One of
the caveats remained however, because, al-
though screens for recessive mutations are
the most promising ones, such screens
are exceedingly difficult and expensive.
Recently, it was shown that a narrowly
focused screening system for recessive
mutations can be highly productive, while
the expenses remained acceptable. More-
over, M. Justice, A. Bradley, and colleagues
made use of a set of strains carrying engi-
neered coat color markers and a chromo-
somal inversion (see Fig. 14a), enabling to
further reduce costs by focusing the screen
on a predefined chromosomal segment.
Given the large number of loci controlling
complex genetic disorders, this strategy
permits an ordered approach to finding
disease genes. The strategy is highly suit-
able to focus on some synthenic regions
in the mouse genome, which are known
to be associated with disease susceptibility
in man.
6 use of mouse strains in cardiovascular
Phenotype Analysis
Genetically engineered mouse models
serve to study genotype–phenotype rela-
tionships and can show an impressive
Transgenic Mice in Biomedical Research 539
variety of phenotypes, ranging from em-
bryonic lethality to increased susceptibility
to chronic diseases such as cardiovascu-
lar disease and cancer. The models allow
defining primary gene functions as well
as the role of a particular gene mutation
in disease.
Once mouse models have been gener-
ated, it is important to carry out an in-depth
analysis of the pathology of the entire
mouse and not restrict the analysis to the
target organs that are the main subject of
the intended study. A detailed strategy is
outlined elsewhere. Often, one will find
that the dramatic alterations in the gene
expression pattern will show unexpected
effects on the phenotype. For instance,
in the case of a study on apolipopro-
tein C1, the expected perturbation of lipid
metabolism was accompanied by a dra-
matic and progressive hair loss of the mice
with the highest level of expression of the
transgene. The most common problem
encountered, however, in studies using ge-
netically modified mouse models is a lack
of phenotype, even when a unique and
evolutionarily conserved gene has been
altered for which there are no obvious
functionally redundant homologs present
in the mouse genome. The main cause
of this is the large difference in environ-
mental stress experienced by wild mice as
opposed to inbred laboratory mice. Often,
phenotypes need to be evoked by either
increasing the environmental stress (i.e.
high-fat diets, mutagens) or increasing the
genetic stress using a second transgenic
strain with a mutation in a related path-
way. A good example is provided by the
research. Wild-type laboratory mice are re-
sistant to develop atherosclerosis because
of its highly antiatherogenic lipoprotein
profile. By knocking out genes control-
ling lipoprotein metabolism, such as the
540 Transgenic Mice in Biomedical Research
apolipoprotein E gene, the mice become
sensitized. Upon feeding a moderated
high-fat diet, such mice will develop com-
plex lesions in the aorta within 2 months.
Many gene alterations will have no ef-
fect on atherogenesis when studied sep-
arately. However, when these mice are
crossed with apolipoprotein E–deficient
mice, it is now possible to study the role
of such genes. Thus, the apolipoprotein
E–deficient mouse serves as a sensitized
mouse model that is highly suscepti-
ble to the effects of additional genetic
changes. Likewise, changing the genetic
background of the mice will affect suscep-
tibility to disease; this approach has been
used to define novel loci in human disease.
Recent technological advances permit
analysis of transgene expression and the
biological consequences thereof by non-
invasive methods. In vital imaging, very
sensitive cameras measure biolumines-
cence from, for example, tumors or em-
bryos in utero, which express luciferase of
GFP reporter transgenes in living animals
(Fig. 2d). Clearly, the impact of technolog-
ical developments like these is substantial
not only in terms of increased specificity
and resolution of the model but also in
terms of reduced animal usage.
7 advantage that they are not silenced in
Perspectives
7.1
Partial Transgenesis; Viral Shuttles
Recent technological advances allow more
rapid approaches to study gene function
without the need for germ-line modifica-
tion. Remarkably, partial transgenesis can
be achieved through local electroporation
of transgenic vectors into tissues. Using
this approach, local muscle tissue in rats
has been specifically modified to study its
function. Another example is the study
of cell migratory and (trans)differentiation
processes during neural tube closure; in
these studies, one side of the tube is se-
lectively electroporated with an expression
construct, while the other side functions
as a control.
The use of viral vectors to deliver trans-
genes to mammalian cells is another
method to achieve partial transgenesis.
The use of several classes and combina-
tions of viral vectors is being explored
in gene therapeutic applications. Similar
vectors are very useful in accomplishing
partial transgenesis, restricted to certain
tissues such as bone marrow (which is
easily harvested and cultured ex vivo and
reintroduced into a recipient mouse). The
latter approach can also be employed to,
for instance, study isolated effects of gene
knockout in the hematopoietic compart-
ment in combination with bone marrow
transplantation. Local and transient trans-
genesis is currently used to activate con-
ditional floxed transgenes (see Sect. 5), for
instance by adenovirus-encoded CRE. This
approach was proven useful in, for ex-
ample, somatic activation of transforming
oncogenes in the lung.
Lentiviral vectors, unlike the classical
slow replicating retroviruses, harbor the
embryonic stem cells, which makes ex-
pression characteristics of such vectors
good. Recombinant lentiviruses were re-
cently used to introduce transgenes into
fertilized oocytes and ES cells (Fig. 6b).
The advantages of using lentiviral shuttle
vector are significant: it obviates poten-
tially harmful procedures otherwise used
to introduce transgenes into a cell (pronu-
clear microinjection, electroporation) and
offers some degree of control over trans-
gene copy numbers.

7.2
RNA Interference; Posttranscriptional
Gene Silencing
The abundance of biological information
that has become available upon completion
of the genome sequence of both mouse and
human is exciting and overwhelming at
the same time. An estimated 10 000 genes
have been identified currently, which rep-
resents an estimated one-third of the total
gene complement in a human or mouse.
Of these genes, only a few thousand have
been functionally studied. Clearly, one of
the remaining challenges in molecular ge-
netics is to analyze the function of novel
genes. Gene targeting is beyond any doubt
one of the most successful and informative
technologies when it comes to elucidat-
ing gene function. Despite improvements
in cloning technologies that employ re-
combination in prokaryotes instead of
‘‘standard’’ recombinant DNA technology
and that significantly simplify otherwise la-
borious cloning strategies, gene targeting
remains time-consuming and expensive.
Recent scientific and technological ad-
vancements may, however, provide faster
means to address novel gene function.
One of the most exciting developments
finds its origin in the realization that
gene expression is controlled at a post-
transcriptional level by double-strand (ds)
RNA-mediated degradation of cytoplas-
mic RNAs. This process, called RNA
silencing – or posttranscriptional gene silenc-
ing – was first discovered in plants as an
unexpected gene silencing in transgenic
plants: silencing of an endogenous gene
often occurred in concert with silencing of
the transgene. Transgene-mediated RNA
silencing is now recognized as a form of
RNA silencing that uses small interfering
dsRNAs (siRNA), which serve as a guide
for specific target mRNA recognition and
Transgenic Mice in Biomedical Research 541
its subsequent enzymatic degradation. In-
vestigations by many groups showed that
RNA interference (RNAi) is a regulatory
process used by many organisms to con-
trol gene expression. Many of the protein
factors involved in RNAi are conserved,
from protozoans to humans. RNAi has re-
cently been developed into a technology
by which endogenous gene function can
be assessed in many vertebrates, includ-
ing mammals. One obvious advantage of
RNAi-mediated gene silencing is that it
does not rely on genetic modification. This
makes the technology very rapid compared
to (conditional) gene targeting. Proof of
principle has been achieved in many ver-
tebrate systems including several human
cells lines. RNAi also works in early mouse
embryos, and was recently demonstrated
to be transmittable via the germline in
transgenic mice. Whereas initial strate-
gies in cell lines made use of synthetic
dsRNA sequences, a relative costly ap-
proach, RNAi, is currently achieved via
stable expression. In addition, recent re-
ports show that random siRNA libraries
can be generated from cDNA libraries via
ingenious cloning strategies. The potential
held by this approach is that such libraries
are self-selecting, thereby enabling inves-
tigators to set-up high-throughput genetic
screens. RNAi can be made tissue specific,
inducible, and can be delivered to mouse
embryos using viral vectors. Such improve-
ments will clearly enhance the application
of RNAi in functional genomic research.
Although genetic manipulation is possi-
ble in tissue culture, the study of genetic
interaction of transgenes with other genes
and local or systemic effects of transgene
expression in the intact organism provides
a much more complete and physiologi-
cally relevant picture of a gene’s function
than can ever be achieved in vitro. Ge-
netic modification therefore represents an
542 Transgenic Mice in Biomedical Research
important and biologically relevant tool
to complement in vitro gene expression
studies aimed at, for example, delin-
eation of signal transduction pathways or
identification of tissue-specific regulatory
elements. Refinements to genetically mod-
ified mouse models in biomedical research
are of importance for a number of rea-
sons. Conditional models recapitulate the
pathophysiology as it occurs in humans
and permit a high degree of selectivity
and resolution, which not only renders
studies more specific and focused but also
significantly reduces the burden on the lab-
oratory animal. The positive effects thereof
are reduced stress on the system and, con-
currently, improved specificity.
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template for gene silencing, Cell 101, 235–238.
Beier, D.R. (2002) ENU mutagenesis: a work in
progress, Physiol. Genomics 11, 111–113.
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