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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 smallest. Mouse husbandry is therefore
easy and economical; its requirements
1.1
for food are modest, and mice are
Mouse Genetics
excellent breeders with a short generation
time, allowing around four successive
The resources directed toward studying
generations each year. Upon completion of
the mouse have been exponentially grow-
the human and mouse genome sequence,
ing. After the completion of the human
the usefulness of the mouse as a model
genome sequence, the mouse was the
animal for the human has been further
second mammalian genome for which reinforced. The number of genes in
the complete sequence was determined. both species is around 30 000. With the
There is no mammalian system that exception of about 200 genes, which
comes close to rival the molecular and were shown unique to mouse or man,
genetic possibilities that the mouse has the vast majority of genes is conserved
to offer to researchers. Many physiolog- between mouse and man. These combined
ical methods that worked fine in larger properties make the mouse an excellent
experimental animals have been minia- animal model to study gene function and
turized to make use of the genetic extrapolate the outcome to human disease.
approaches uniquely available to mouse Second, the mouse has advantages that
researchers. What makes the mouse may be less obvious, but which relate to the
so special? long tradition of mouse research starting
The advantages that come to mind first in the early 1900s. Up until then, hobbyist
are obvious: for biomedical researchers, mouse keepers had already been breeding
mammals are highly preferred model mice for their coat color variation. By
systems, because the physiology of man making use of these mice, it was possible
and most mammals is very similar and the to demonstrate that this variation followed
pathology of most diseases is reproduced the laws of Mendelian inheritance. Soon, it
well in animal models. The mouse stands was also realized that there was a need for
Transgenic Mice in Biomedical Research 509

genetically homogeneous mouse strains of another strain. Along these lines, a


and the most widely used inbred strains very powerful resource has been devel-
such as BALB/c and C57BL6 originate oped recently, consisting of a panel of
from that period. An excellent historical inbred mouse strains carrying only a sin-
overview on mouse genetics is provided by gle chromosome from the other strain
Dr. Silver. (http://jaxmice.jax.org/library/notes/
At present, hundreds of inbred strains 493c.html). These chromosome substitution
have been generated (see Sect. 6). This re- strains are very similar to recombinant
source is the key to the success of mouse inbred strains, but offer a less costly strat-
research. Inbred strains ensure that exper- egy to move from a susceptibility locus
iments can be carried out in the absence of toward the gene proper. The principles of
inter-individual genetic variation, because mouse genetics are crucial when working
mice within an inbred strain are all geneti- with transgenic animals, because it is vi-
cally identical to each other and homozygous tal to control the genetic background in
throughout the genome. Moreover, each the experiments (see below): the interac-
strain has one or more unique and, often, tion between the transgene and the genetic
well-established properties that make the background will ultimately determine the
strain particularly useful for a specific area phenotype; for example, changing the ge-
of research. For instance, some mouse
netic background on a given genetic trait
strains appeared susceptible to cancer or
may prevent an early death of a particular
cardiovascular diseases, while others are
mouse model.
better suitable for behavioral research. The
While mouse genetics has a major
genetics of such complex diseases and bio-
impact on our understanding of the mech-
logical processes is very difficult to study
anisms underlying complex diseases, a
in man. This is mainly due to the fact
constant influx of mice carrying single
that multiple genes are involved and that
gene mutations greatly stimulates mouse
the genetic basis for susceptibility to, for
instance, cancer will vary from person to research. These mutations have either oc-
person. In the mouse, it is possible to cross- curred spontaneously in mouse breeding
breed a susceptible and a resistant strain. programs or originated from research on
As a result, the susceptibility to cancer will the gene toxicity of radiation and chem-
vary in the progeny. Genetic variation in ical compounds. The latter approach has
these mice is confined to the differences been rejuvenated upon the availability of
between the two parental strains, and ev- better technology to identify the causative
ery given locus will only have two different mutations (see below). A good example
alleles. Hence, the genetics is confined of a spontaneous mutation is the lep-
to a maximum of two alleles per locus, tin gene mutation that occurred in the
which greatly simplifies genetic analysis. C57BL6 mouse. The mutation was named
These properties allowed generating spe- ob, after obese, because the mice have no
cific resources to study complex diseases, control over their feeding behavior and be-
including recombinant inbred (RI) strains come overwhelmingly obese. Hence, the
(http://jaxmice.jax.org/info/recombin- phenotype could be discovered easily. The
bred.html). These RI strains combine subsequent discovery of the causative gene
a specific genetic contribution of one marks one of the most dramatic discover-
mouse strain with the genetic background 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 and vertebrate species, including mam-
combination of technologies offers the pos- mals such as mice, rats, rabbits, and
sibility to design models with ‘‘gain of livestock such as sheep, pigs, cattle, and
function’’ and ‘‘loss of function,’’ which is recently even primates. The choice of the
an ideal situation for a (mouse) geneticist models is primarily determined by its ob-
(Fig. 1a). jective (e.g. improvement of economical
Notably, these technological break- value or development of pharmaceutical
throughs were facilitated by the availability proteins). Although fundamental biolog-
of inbred mouse strains. Transgenesis ical processes can be well studied in
works most efficient when the donor eggs relatively simple organisms such as the
are produced by first generation (F1) hy- nematode Caenorhabditis elegans, or the
brids between C57Bl6 and BALB/c. In the fruitfly Drosophila melanogaster, for experi-
case of the gene-targeting approach us- mental biomedical research the laboratory
ing embryonic stem cells, it is critical to mouse is by far the most used animal
make use of the appropriate strain com- model. Below, we will briefly outline the
binations when injecting the cells into emergence of the two technologies and
blastocysts and propagating the embryos sketch the initial experimental strategies
in foster strains. This dependence on the that caused great excitement within the
genetic background provides the ability to scientific community.
choose the optimal strain and may be one
of the reasons why, mice are, as of yet, 1.2.1 Brief Overview of Developments
the only mammalian species suitable for in Transgenesis
gene targeting (see Sect. 3). The goal of By classical definition, a transgenic organ-
altering the gene function through trans- ism carries extra, often foreign (i.e. from
genesis is to generate animal models in a different organism) DNA in its genome,
which the role(s) of the gene of interest called a transgene. Transgenesis literally
either in normal (e.g. development) or ab- crosses (trans) species barriers. One of the
normal biological processes (e.g. disease) most obvious conceptual characteristics
is revealed. Other important applications is that a transgene integrates at random
in fundamental science include the study in the recipient host genome, whereas
of mammalian gene expression regula- gene targeting, by homologous recombi-
tion. Currently, genetic modification is nation, selectively alters an existent locus.
carried out in a wide range of invertebrate 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 the complete intron–exon structure of the
of modern-day mouse molecular genetics, genes, and including large up- and down-
are summarized in Fig. 1(b). stream DNA segments. Such fragments
In the mid 1970, robust technology to were obtained through cloning in cosmids,
clone genes and manipulate genes was bacterial artificial chromosomes and yeast
still about five years away. Subsequently, artificial chromosomes, allowing handling
as the structure of eukaryotic genes was re- DNA fragments in the range from 40 up
vealed and recombinant DNA technology to 1000 kb.
developed, gene constructs could be gen-
erated comprising an eukaryotic promoter 1.2.2 Brief Overview of Developments
fused to the full-length coding sequence of in Gene Targeting
the gene of interest (consisting of a DNA The development of gene targeting by
copy (cDNA) of the mRNA molecule). homologous recombination in embryonic
Generating mice with a strong promoter stem cells (ES) can be attributed to two cru-
driving an extra copy of the gene resulted cial technological advances that ultimately
in mouse models with high-level expres- compounded. One line of research in-
sion of the gene of interest. Interesting volves ES research. Initially, ES cells were
phenotypes did occur when the gene was used to study cellular differentiation and
markedly overexpressed. In addition, how- cancer, because when injected subcuta-
ever, genes would become expressed in neously into nude mice, teratomas would
the wrong tissue or at the wrong place. develop. Figure 1(b) itemizes a number
This ectopic expression was also a powerful
of crucial advances in ES cell technology
way to obtain phenotypic information. Ini-
that helped develop ES cell technology
tially, researchers were often confronted
to its current status. Improved culturing
with a high failure rate: gene constructs
conditions ensured sustenance of pluripo-
would be integrated in high-copy head-
tence, a property of ES cells crucial for
to-tail arrays (see Sect. 2), but would not
their application as vehicles to introduce
show a corresponding increase in expres-
germ-line mutations. Soon after the first
sion level, or transgene expression would
diminish in consecutive generations. A mouse was made that showed germ-line
much-improved understanding of eukary- transmission of genes introduced through
otic gene regulation in the mid-1980s genetic modification of ES cells in vitro, evi-
provided the insight, leading to improved dence was obtained that an endogenous
construct design. For instance, DNA ele- locus could be exchanged for a mutated
ments located tens of kilobases outside of sequence by homologous recombination
the β-globin gene were shown to control in vitro (see Sect. 3). These strategies tar-
high-level tissue-specific gene expression geted the murine HPRT gene and were
through interaction with DNA sequences based on the fact that HPRT is an ex-
immediately 5 -prime of the gene. Also, cellent selectable marker expressed in
sequence elements were discovered that the genome on the X chromosome of
isolate a specific gene from influences male cells. Spurred by success, the ques-
of flanking DNA. In addition, evidence tion arose whether gene targeting would
was obtained for a role of splicing in also be feasible for nonselectable auto-
mRNA stability. These advances led to the somal genes; this was proven possible
use of much larger constructs harboring shortly after.
Transgenic Mice in Biomedical Research 513

Sections 2 and 3 will elaborate on organisms such as yeast, fireflies, or


the conventional transgenesis and gene- jellyfish, which reveal their presence by a
targeting technology. Noteworthy, with the microscopically or macroscopically clearly
introduction of novel concepts in molec- visible mark (Fig. 2a, b). This approach is
ular genetics, our ability to manipulate standardly used to examine spatio-temporal
gene expression in the mouse currently gene expression patterns and/or tissue
knows few limitations. Sections 4, 5, and specificity of native promoter sequences
7 of this chapter will elaborate on some in vivo (Fig. 2b).
of the more recent technological advances By far, transgenesis is used to achieve
such as binary systems (see Sect. 4), ran- and/or study the effects of overexpres-
dom mutagenesis (see Sect. 5), and RNA sion (higher gene activity than normal)
interference (see Sect. 7). or ectopic expression (gene activity in dif-
ferent cell types than normal) of a gene
of interest, or of a mutated form thereof.
2 The common denominator of these stud-
Transgenesis; Basics and Technology ies is the analysis of the resulting altered
phenotype. This can be abnormal devel-
2.1 opment, altered physiology or behavior,
Transgene Design development of disease, and so on. The
choice of regulatory sequences (see be-
Shortly after its introduction in the early low) determines whether the expression
1980s, the main scientific applications of a transgene follows the expression pat-
of transgenesis in the mouse could be tern of its endogenous counterpart or is
roughly divided in two areas: (1) studies limited to distinct cell types or particular
of gene promoter activity (and/or other developmental stages.
regulatory elements) and (2) studies of
the effect(s) of gene overexpression (or 2.1.1 Origin and Structure
a mutated form thereof; Fig. 2a). Gene of the Transgene
activity is, simply put, controlled by a The origin of a transgene, or elements
promoter. A promoter is a stretch of therein, may range from prokaryotes or
DNA sequence, which usually precedes the from lower (e.g. reporter genes) to higher
coding part of a gene. A promoters’ task is eukaryotes, like mice or men. In ex-
to regulate gene expression: it controls the perimental biomedical research, human
‘‘when (at which time point) and where’’ transgenes offer several advantages. Im-
(in what cells, tissues) of gene activity. portantly, many known genetic disorders
Eukaryotic genes also carry a termination in humans have been extensively char-
element. In order to function properly acterized at the molecular genetic level:
in a eukaryotic genome, a transgene mutations or deletions are charted and
minimally meets these requirements: it such ‘‘diseased alleles’’ are readily avail-
carries a promoter, a coding part, and a able. In addition, most genes and proteins
stop signal. The easiest way to read out are well conserved between mouse and
promoter activity in cell or a developing man. This, of course, is important when
animal is by placing a so-called reporter the biological activity of a gene product de-
gene downstream of it. Reporter genes pends on proper interaction with other
encode proteins, often found in lower cellular proteins. Finally, the sequence
514 Transgenic Mice in Biomedical Research

divergence between mouse (transgene re- preserved in a transgene construct. Solely


cipient) and human (transgene donor) of cDNA-based expression vectors (i.e. exons
a given gene can be used to determine sequences only) frequently show low ex-
whether a mouse is transgenic or not, what pression levels and are often silenced.
its copy number is (number of transgene Since many integrating DNA or RNA
integrations), and whether a transgene is viruses do not carry the eukaryotic in-
actively expressed, since it allows for dis- tron/exon structures, an expressed genetic
crimination between transgene and the sequence without such boundaries may
endogenous gene. Figure 3 gives an exam- be recognized as foreign and potentially
ple of such an analysis. harmful by the eukaryotic host cell and
Most eukaryotic genes display a typi- will be inactivated (silenced) by cellular de-
cal intron/exon structure, with only exon fense mechanisms. The native intron/exon
sequences represented in mRNA. Trans- structure of a gene need not be preserved
genes, in general, are more reliably ex- in its entirety: indeed, inclusion of only
pressed if the intron/exon boundaries are one intron in a transgene has been shown

Construct type

1. Promoter X Reporter gene Promoter studies

2. Promoter Gene X Gene studies


(a)

(b) (c)

(d) (e)
Transgenic Mice in Biomedical Research 515

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


successful transgene constructs include fold attachment regions (SARs), Locus
both genomic and cDNA sequences de- control regions (LCR), chromosomal insu-
rived form the same gene (Fig. 4a). lators, and antirepressor elements. Some
of these elements are involved in sub-
2.1.2 Regulatory Elements nuclear localization of genes to areas of
Any transgene comprises a number of active transcription and/or insulate gene
essential elements that control gene ex- expression from influences of surround-
pression (Fig. 4b). The most basic and ing chromatin. Some of these autologous
essential elements are the promoter and (i.e. endogenous) regulatory elements may
termination signals for transcription (RNA actually be many thousands of base pairs
synthesis). A promoter controls gene ex- removed from the coding sequences of a
pression by providing binding sites for gene. Indeed, if the purpose of a study is
proteins (RNA polymerase transcriptional to examine faithful gene expression pat-
machinery, cell type–specific transcription terns, for instance throughout embryonic
factors) that regulate gene transcription development, a transgene should include
(i.e. activation). The real-life situation is such endogenous elements. The exact lo-
often much more complicated than this; cation of such elements within a locus
many more regulatory elements exist that is often not known and hence there is an
all play a role in establishing stable and obvious advantage in using large DNA seg-
faithful gene expression patterns. Among ments as transgenes. The development of
these are enhancers, which typically act molecular vectors, which can harbor large
in an orientation-independent manner, 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; pression or ectopic expression is required,
PACs), or bacteria (BACs) has been im- general type heterologous promoters (e.g.
perative for the cloning and expression viral promoters) and/or enhancers are
of such transgenic constructs. Exceedingly widely used. The use of heterologous
large constructs (>500 000 bp) are first and autologous regulatory elements is
transferred into embryonic stem cells (see often combined. The first published trans-
Sect. 3), which are then used to generate genic mouse model displaying a specific
transgenic mice. phenotype made use of the general-type
Regulatory elements need not necessar- metallothionein promoter (pMT). The MT
ily be autologous in nature, they may be 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 the animal model (see Fig. 2). Not only
bacterial endotoxin (LPS). Heterologous general-type promoters, like the ones driv-
promoters and other regulatory elements ing housekeeping genes (e.g. phosphoglyc-
are applied widely, and as indicated be- erate kinase PGK) or other genes that are
fore, their incorporation into constructs widely expressed throughout eukaryotic
is determined primarily by the aim of cell types (e.g. histones, ß-actin), but also
518 Transgenic Mice in Biomedical Research

viral promoters are often used to ensure of a foreign DNA sequence in the mouse
high and ubiquitous transgene expression. was achieved through retroviral transduc-
LCRs are included in transgenic constructs tion. As indicated above, viruses of this
to both enhance transgene expression class are often inactivated in eukaryotic
and to achieve position-independent and cells. Although recent technological de-
copy number–dependent expression of a velopments in the field of transgenesis
transgene, often with cell lineage–specific again make use of retroviral constructs (see
enhancer activity. Finally, promoter choice Sect. 7), pronuclear injection has been the
is often dictated by whether expression of golden standard for generating transgenic
a transgene is deleterious to the mouse or animals over the last two decades.
not. For example, many oncogenes play an The principle of pronuclear injection
important role in embryonic development, is simple: a sterile DNA solution is pre-
interfering with their normal expression pared, which contains a very low con-
(pattern) results in embryonic lethality. To centration of the transgene (nanograms
bypass this problem, either tissue-specific (10e-12 kg)/microliter (10e-15 l)). A very
or inducible promoters (or combinations fine glass needle containing the transgene
of such regulatory systems are used (see
DNA solution is inserted into one of the
Sect. 4)).
pronuclei and DNA solution is expelled
Taken together, the origin of DNA, struc-
into the nucleoplasma (Fig. 5a, 6a). The
ture and choice of regulatory elements
amount of DNA injected is extremely low
for a transgene are largely determined
(femtograms (10e-18 kg)/picoliters (10e-
by the scientific aim of the experimental
12 l)). Injected transgene DNA in the nu-
model itself. For biomedical studies em-
cleus tends to integrate at random into the
ploying the laboratory mouse as a model
genome. Often, this occurs in head-to-tail
system, the use of human transgenes is
often preferred. tandem arrays called concatamers. (Fig. 5b).
The repetitive nature of such concatamers
2.2
may lead to transgene inactivation, as does
Pronuclear Injection the random character of the insertion: it
often causes transgenes to land in inactive
Transgenes can be introduced into living heterochromatin, which constitutes most
cells in many different ways. Among the of a eukaryote cells’ DNA. The use of in-
most standard procedures in vitro (‘‘in the sulator elements (see Sect. 2.1) or targeted
test tube’’) are transfection methods either transgenesis (see Sect. 4) provides solu-
using calcium phosphate/DNA precipi- tions to these problems.
tates or liposomes (small DNA-containing
vesicles surrounded by a lipid membrane), 2.3
which are taken up by cells, electropo- Biotechnological Procedures
ration (electroshock), viral vectors (e.g.
adenovirus, retrovirus, which carry the Figure 6 gives an overview of the biotech-
transgene), particle bombardment, and nological procedures involved in the pro-
microinjection. Some of these methods, duction of transgenic mice. Because gen-
however, cannot be used in combination erating transgenic mice by pronuclear
with mouse oocytes. The very first proof-of- injection is a rather inefficient process, a
principle report on germ-line transmission 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.
Transgenic Mice in Biomedical Research 521

Convenient numbers of fertilized eggs transgenesis asks for genes and their phe-
are obtained by superovulation, much the notypes to be dominant in nature (e.g.
same as way as in humans for in vitro fer- expression of (mutated) oncogenes), gene
tilization purposes: exogenous hormones targeting now permits study of reces-
(follicle-stimulating hormone and luteiniz- sive gene function (e.g. tumor suppressor
ing hormone analogs) are injected at a genes). The concept of gene targeting is
48-h interval to achieve multiple ovulations simple: eradication of a gene may reveal its
per donor female, which are subsequently function by a resultant phenotype. Current
mated to fertile males. Simultaneously, applications range from conventional null
recipient females, the so-called foster fe- mutation of a given gene, the so-called gene
males, are endocrinologically primed for knockouts, to replacement of endogenous
pregnancy by copulation to vasectomized alleles with mutated alleles and even in-
males (see Fig. 7). Microsurgery is carried ducible (conditional; see Sect. 4.3) genetic
out under aseptic conditions and general modifications.
anesthesia. A number of different mouse
3.1
strains are useful for biotechnological pro- Targeting Vector Design
cedures, many of which have their own
advantages and specific traits (see Sect. 1). Generally speaking, two types of vectors
Detailed descriptions of the technology, exist by which genes can be mutated.
useful strains, and husbandry are to be
found in a number of media. 1. Replacement vectors
2. Random insertion vectors
3
All classical replacement type vectors have
Gene Targeting; Basics and Technology
in common that they carry stretches of
DNA, which are highly homologous or
First established in the late 1980s and early
identical to the gene, which is to be tar-
1990s of the last century, gene targeting is
geted. The mechanism by which gene
now a well-established technology for gen-
replacement is accomplished, homologous
erating genetically modified mouse mod-
recombination, uses these identical or very
els. Gene targeting allows for germ-line similar sequences of DNA: homologous
manipulation at predetermined genomic recombination is a sequence homology-
loci by a process called homologous recom- initiated and driven process (Fig. 5c, 8a).
bination: part of an endogenous allele is It appears to be a highly selective pro-
replaced or mutated by vector sequences cess, which only takes place between
that carry flanking sequences homologous relatively large (several kilobase pairs)
to the endogenous gene (Sect. 3.1). It is DNA segments. In fact, it is important
in this aspect where gene targeting funda- that the constructs used for homologous
mentally differs from conventional trans- recombination are being derived from
genic technology: the possibility to change the exact same genetic background (iso-
existing genes offers a very important and genic) as the cell line used for gene
powerful extension of the molecular ge- targeting. This requirement implies that
netic tools to create experimental animal a sequence divergence of around 1 in
models in biomedical research. Whereas 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.
Transgenic Mice in Biomedical Research 523

the frequency of homologous recombi- sequences, thereby interrupting the gene


nation. Conventional gene-targeting con- and rendering it nonfunctional (Fig. 8a).
structs mostly harbor a selection marker In addition, the marker is used to positively
(neomycin, hygromycin, or puromycin re- select for embryonal stem cells that have
sistance gene), which serves a number integrated the targeting vector. A second
of purposes: it replaces endogenous gene feature often included in a replacement

Functional domain

Protein Gene product

R R

Probe Locus

Homologous
recombination

R R

Select Targeting vector

(a) Interruption & selection

Genotype: +/+ −/− +/−


(R-R fragments) Wild-type allele

Null allele
(b)

Fig. 8 Basic design of a conventional targeting that by means of an ‘‘external probe’’ (blue-grey
vector. (a) The targeting vector duplicates part of box: probe; from within the to be targeted locus,
the locus to be mutated (homologous but outside the homology regions used in the
sequences), but is interrupted by a positive targeting construct) and strategically chosen
selection marker (select), for instance, an restriction endonuclease sites (R) a shift is
antibiotic resistance gene. Many conventional detected upon homologous recombination. The
targeting vectors also contain a negative figure shows the three possible genotypes; two
selection marker. The herpes simplex virus
wild-type (+/+) alleles, two knockout (−/−)
Thymidine Kinase gene is often included: the TK
alleles (homozygous knockout), and a
gene product converts a harmless compound,
for instance, Ganciclovir, into a cytotoxic combination of both in the heterozygous (+/−)
substance. The positioning of the TK cassette at condition (see also Fig. 10). Mutagenesis vectors
one of the ends of a targeting vector results in may be introduced into ES cells via several
exclusion of TK from the genome upon means; the most commonly used method is
homologous recombination, whereas TK is electroporation (electroshock) by which the
cointegrated in case of random integration of a cellular membrane is damaged, allowing uptake
targeting vector. (b) The vector is designed such of DNA in cells (see color plate p. xxvii).
524 Transgenic Mice in Biomedical Research

vector is a negative selection marker, for ex- on a layer of supporting feeder cells (mouse
ample, the Herpes simplex virus thymidine embryo fibroblasts (MEFs)) in the presence
kinase (HSV-TK). Upon positive (negative) of factors that block differentiation of stem
selection, a number of separately isolated cells in general (like Leukemia Inhibitory
ES cell clones are ‘‘genotyped’’ to ensure Factor,) or are ‘‘feeder-independent.’’ It
proper recombination on both ends of the is their pluripotency that is ultimately
targeting vector. Typically, these cells carry called upon: when generating targeted mu-
a monoallelic mutation, that is, only one tations in the mouse, male mutant ES
of two alleles in the ES clone is replaced by cells are reintroduced into preimplanta-
the targeting vector (Fig. 8b). tion embryos and made to participate in
Random insertion type vectors insert at embryogenesis and spermatogenesis in
will in the genome and, therefore, do particular. If the latter occurs, the tar-
not rely on sequence homology with geted mutation can be stably transmitted
target genes (see Sect. 5.1). Integration via the germline to offspring and bred to
of a vector in a locus may, however, homozygocity.
cause inactivation of a gene. The best-
known application of insertion type vectors 3.3
is gene trapping (see Sect. 4 and 5). Genesis of Mosaic Embryos
Gene trap vectors are constructed such
that they either trap promoter activity ES cells may be used in a number of ways to
or trap a polyadenylation signal from generate genetically mosaic embryos. The
an endogenous locus (Fig. 15). In this most commonly used procedure is blasto-
manner, not only actively transcribed cyst injection: preimplantation blastocysts
but also inactive genes can be trapped are used to introduce ES cell into (Fig. 5a).
(with promoter-trap or poly(A)-trap vector The donor mouse line (i.e. of which ES
respectively). Variations on this concept cells are derived) and the recipient line (i.e.
are described in Sect. 5. of which blastocysts are procured) differ in
coat color genetics. Typically, an inbred
3.2 strain such as C57blk (black coat color)
Embryonic Stem (ES) Cells is used for blastocyst production to in-
ject 129Sv or a 129Ola (sand-colored coat),
In contrast to classical transgenesis, where some of the most commonly used ES cell
mutagenesis is done directly in the em- lines, into. The procedure described above
bryo, gene targeting is done in embryonic is schematically represented in Fig. 6c.
stem cells (ES cells) (Fig. 9a). These are very Alternatively, ES cells can be fused to
early stem cells isolated from preimplan- morula stage embryos in a procedure
tation embryos (blastocysts). ES cells are termed morula aggregation. Embryos in the
pluripotent, that is, they are fully undiffer- morula stage typically comprise 8 to 16
entiated and, in essence, have the capacity blastomeres (individual embryonic cells)
to participate in mouse embryogenesis and surrounded by a protective layer called
become any cell type in the animal. The the zona pellucida. In order to achieve
first steps in a gene targeting experiment aggregation, this zone is first removed and
are carried out in vitro. ES cell culturing the embryos together with a small number
conditions are aimed at maintaining their of ES cells are cocultured in a small
pluripotency. ES cells are either cultured depression, in which gravity forces them to
Transgenic Mice in Biomedical Research 525

(a) (b)

(c) (d)

Fig. 9 Embryonic stem cells, embryos, and cells – sand) becomes manifest as Agouti,
chimeric mice. (a) Left panel: ES cells grown on patches of purely 129Ola-derived tissue is sand
mouse embryonic fibroblasts (MEFs); white colored (e.g. over left eye). (d) Electrofusion of
arrows point at colonies of tightly coherent ES two-cell-stage embryos (middle left) results in
cells; right panel: feeder-free ES cells. fusion and formation of one-cell-stage tetraploid
(b) Aggregation of 8-cell-stage morulas (black embryos (white arrows). Tetraploidy does not
arrow) with genetically modified ES cells (white hamper placenta formation, but does not permit
arrow). (c) Chimeric mice beautifully displaying normal embryogenesis. Tetraploid blastocysts
the coat color mosaicism resulting from injected with diploid ES cells will support fully ES
contribution of ES cells of two different genetic cell–derived embryonic development (source
backgrounds to the developing embryo the mix image: (http://www.mshri.on.ca/nagy/cre.htm))
of, in this case, C57Bl (recipient (see color plate p. xxxi).
blastocyst – black) and 129Ola (donor ES

be in close contact with each other (Fig. 9b; the coat color mosaicism are referred to as
http://www.mshri.on.ca/nagy/cre.htm). chimeric mice (Fig. 9c). Chimeric males are
Both methods yield the typical mosaic bred to wild-type mice to generate a mouse
coat (e.g. a mixture of different coat colors, that is heterozygous for the mutant allele in
like black and sand). The mice that display all its cells; the subsequent mating of two
526 Transgenic Mice in Biomedical Research

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

4.1
4 Fully ES Cell–derived Embryos
Refinements to the Models
The placenta is one of the first organ
Transgenes or gene knockouts may cause systems to develop during early embryo-
unexpected embryonic or neonatal lethal- genesis. It provides an essential means
ity, either by interfering with embryo by which the developing embryo and the
development or placentation. If the aim mother animal exchange essential factors
of an animal model is to, for example, such as nutrients and oxygen. A surpris-
ingly large number of null mutations result
study the effect of a transgene or a gene
in defective placentation. Defective placen-
knockout on adult physiology or devel-
tation in a conventional genetic approach
opment of a disease, embryonic lethality
thwarts all possibilities to uncover a gene
limits the usefulness of such a model. To
function during early embryogenesis. J.
circumvent this problem, animal molecu-
Rossant and coworkers developed an ele-
lar geneticists have sought to refine animal gant solution to this problem during the
models. The resulting mouse models of- early 1990s: tetraploid embryos were used
ten combine aspects of transgenesis and to reintroduce diploid ES cells into. The
gene targeting and may include a molec- concept is elegantly simple: tetraploid cells
ular switch, by which null mutation of a will contribute to placentation (polyploidy
gene now is rendered conditional. Such is a normal feature within the developing
on/off models are referred to as binary placenta); they will, however, not con-
models. The most pivotal advantages of tribute to the development of the embryo
such molecular switches are gain in ac- itself. It was shown that in this approach,
curacy and specificity of the model and reintroduced normal diploid wild-type ES
an important concomitant reduction of cells were fully capable of supporting nor-
disease burden for the animals. Some ex- mal embryonic development. The data
citing models and applications that have 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, bryo (Fig. 9d). This tetraploid early embryo
while, at the same time, potential negative is allowed to further develop in vitro, to be
effects of a null mutation on placental de- used at a later stage for aggregation or
velopment were circumvented. Tetraploid microinjection (see Sect. 3.3). It should be
recipient embryos were generated by elec- noted that although the role of a gene
trofusion: normal diploid two-cell-stage can now be studied independent of pla-
embryos were subjected to a defined elec- centation, specific lethality as a result of
troshock that results in fusion of the cells 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- tissue or select cells within a tissue, for
els are used. instance, epithelial lung cells (surfactant
protein promoter) or anterior pituitary
4.2 cells (POMC, pro-opiomelanocortin pro-
Conditional Transgenesis moter). Several tens to hundreds of mod-
els have been created and used in this
Conditional transgenesis is employed to
fashion to successfully study the specific
have better control over (trans)gene ex-
effect(s) of transgene expression in devel-
pression in a given model. This may be
opment or disease.
advantageous for a number of reasons, for
Truly conditional transgenesis, in which
instance, to circumvent lethality during
embryonic development caused by toxic or the investigator not only determines where
teratogenic properties of transgenes or to a transgene is turned on but also when
study the onset of disease from its earliest and how strong, can be achieved with bi-
stages onward. nary models. Binary models incorporate
In its simplest form, conditionality is in- a switching mechanism. Several molec-
tegrated in an animal model by means of ular switches exist to turn on or off
tissue-specific promoters and/or control transgenes. Figure 11 lists examples of
element usage (see Sect. 2.1). Transgene these methods. Most binary switches
expression is confined to a defined target 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 since mammalian cells lack insect hor-
promoters and transcription factors and mone receptors, the binary system
(2) integration of transcriptional interrup- theoretically works as an on/off system,
tions that are removed by recombination. much like the Tet-system. Ecdysone
The use of inducible transcription by nor its derivatives are toxic or terato-
means of nonmammalian, for example, genic. Owing to excellent pharmacoki-
regulatory elements borrowed from bacte- netic properties, the method is ideally
ria or lower eukaryotes, like the fruit fly (D. suited for very precise conditional con-
melanogaster), allows transgenes to be ac- trol over gene expression.
tivated and silenced ‘‘on command.’’ The – Transgene induction by removal of a tran-
system that utilizes removal of a strong scriptional block. The system makes
transcriptional block by means of recombi- use of a recombination process de-
nation, although in principal reversible, is, rived from bacteriophage P1: phages
for practical reasons, mostly used one-way. (bacterial viruses) use a site-specific
Several binary switches are item- recombinase (CRE) to integrate their
ized below: DNA into the genome of their bacterial
– Tetracycline-controlled transgene expres- host. An essential second component
sion: the Tet-system. The transgene of of this process is a recombinase recog-
interest is placed under the control of nition site, called an LoxP site: two LoxP
a Tetracycline resistance operon from sites are recognized and recombined by
Escherichia coli, the tet-operator (tetO). CRE (Fig. 12a). The two essential com-
A second, independent transgenic line ponents, LoxP and CRE, also work in
expressing a derivative of TetR, a tran- eukaryotic systems, and are commonly
scriptional regulator of tetO, confers maintained on two separate transgenic
Tetracycline (Tc)-inducibility or repres- lines. Flanking a sequence with two
siveness to the transgene of interest similarly oriented LoxP sites will lead
(see Fig. 11a). Transcription factors to its excision in the presence of CRE.
with regulatory activity toward the Hence, an on/off transgenic system
tetO are absent in eukaryotic cells, employs a silenced transgene, in which
which means that theoretically the sys- a strong transcriptional block is brack-
tem cannot be leaky for transcription eted by two similarly oriented LoxP
and pleiotropic effects are therefore sites; a second independent transgenic
not expected. line, which expresses the CRE recom-
– Insect hormone–controlled transgene ex- binase, may be driven off a general,
pression. The system uses the molting tissue-specific or inducible promoter,
steroid hormone ecdysone for transgene depending on the objective of the
induction. It requires a heterodimeric study (Fig. 11b). More details on this
receptor and an ecdysone responsive recombination-based switching mech-
element, which is incorporated as a reg- anism will be discussed in Sect. 4.3.
ulatory element in the transgene of in-
terest. Administration of ecdysone (or a 4.3
derivative muristerone A) rapidly and se- Conditional Gene Targeting
lectively induces transgene expression.
Upon withdrawal of the steroid hor- Gene targeting is widely used to study
mone, expression is silenced. Again, 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 and degree of regulation of the condi-
the conventional approach, in which the tional knockout. CRE-transgene expres-
null mutation is transmitted through the sion is controlled either by constitutively
germline, as discussed in Sect. 3.1. An cell type–specific or inducible regulatory
estimated one in three null mutations elements (also see Sect. 2.1 and 4.2).
results in embryonic or early postpartum Deleter strains are most often generated
lethality. Therefore, germ-line mutations by conventional transgenic technology. Al-
may be appropriate models to recapitulate ternatively, the CRE-cDNA can be inserted
human congenital genetic disorders. They via targeted mutagenesis to a locus of in-
may, however, not be the best tools to terest, under control of the endogenous
study gene function in adult mice, since promoter. Although the latter method
this is hampered by often unexpected and does not require a thorough knowledge
unexplained lethality. K. Rajewsky and of the regulatory elements controlling the
coworkers were among the first to develop
locus at hand, the approach is not as
a conditional gene-targeting strategy that
straightforward as conventional transge-
allows inactivation of a gene in a defined
nesis. Drawbacks of the former method
spatio-temporal setting: the null mutation
comprise all known problems linked to
can, for instance, be confined to selected
conventional transgenesis: transgene ex-
cell types or to a specific stage during
pression is dependent on integration site
development.
Conditional gene targeting makes use and copy number. Consequently, a num-
of the CRE-LoxP principle, as described ber of independent founders will have to be
in Sect. 4.2. Briefly, LoxP sites flank a generated to select a useful strain. A sub-
genomic sequence in a targeting vector, stantial collection of tissue-specific CRE
which will be deleted in vivo; this allele mouse lines is currently available.
is referred to as a floxed allele (Fig. 12b). Inducible CRE expression allows for
This final ‘‘floxed’’ wild-type allele only a high level of control (see Sect. 4.2).
carries recombinase recognition sites at Again, it should be noted that conditional
positions within the locus where they are gene targeting is mainly used unidirec-
presumed inert, for example, introns: up tionally, that is, once a gene of interest is
until the moment of recombination-driven inactivated through CRE-mediated recom-
excision in vivo, the allele should function bination, reversion of the genotype is not
as a wild-type allele. Since the CRE-LoxP easily accomplished.
system is applied more widely than the
FLP-frt system in vivo, further descriptions
of conditional gene targeting will focus 4.3.1 Controlled CRE Expression
mainly on the CRE-LoxP system. Inducibility can be conferred by rather
The current standard experimental con- simple measures such as CRE expression
ditional approach requires two separate through infectious viral particles. Such
mouse lines: viruses can be locally administered or
systemically. In addition, their tropism (i.e.
1. the ‘‘floxed’’ line (as described above)
host-cell range) can be manipulated.
2. the ‘‘deleter’’ line, expressing CRE.
Inducible mammalian promoters have
The transcriptional control directing been used, as well as the Tet-system (see
CRE expression determines the specificity Sect. 4.2). Besides the induction of CRE
Transgenic Mice in Biomedical Research 533

transcription and translation, CRE induc- address such issues. CRE-mediated re-
tion has also been achieved at the post- combination has provided possibilities and
translational level. This was accomplished strategies to manipulate the genome be-
by fusing CRE-encoding sequences in yond conditional gene knockouts or trans-
frame to the ligand-binding domain (LBD) genesis. As illustrated in Fig. 13 and 14,
of nuclear steroid hormone receptors, such the CRE-LoxP system can be used to ma-
as the estrogen or progesterone recep- nipulate the genome in various manners:
tor (ER, PR respectively). Ligand binding
transfers the CRE-ER fusion product into – Gene knockin or gene exchange strategies
the nucleus, where the recombinase exerts apply CRE-LoxP-mediated recombina-
its catalytic activity toward LoxP sites. Mu- tion to exchange endogenous coding
tation of the ER moiety, has generated regions for exogenous or mutated se-
CRE-MER (mutated estrogen receptors) quences in a locus (Fig. 13a). Espe-
fusions, which are only induced by syn- cially, the latter application is of signif-
thetic hormones and not by their natural icant interest to the scientific commu-
ligands (Fig. 11c). Ongoing improvements nity, since it permits the development
of these and alike inducible systems clearly of animal models, which reflect hu-
generate a highly sensitive and specific man genetic disorders more closely
means to control transgene expression and than models applying overexpression
enable the study of gene ablation and or ablation of a gene. Initial ap-
overexpression that, without such tools, proaches inserted reporter genes into
were impossible. An increasing number an endogenous locus; this simple vari-
of published models employing CRE-MER ation on conventional gene targeting
variants attest to the feasibility of the ap- achieves the same purpose as a trans-
proach in vivo. gene harboring a reporter preceded
by endogenous regulatory sequences
4.4 (see Sect. 2.1). The main difference, of
Knockins: Humanized Mice course, is that this strategy obviates
the handling of large DNA fragments
Many studies in human disease have un- with potentially unidentified regulatory
covered mutations that are relevant to elements needed to achieve faithful ex-
the etiology of the pathological condition. pression patterns, since these elements
Examples of these are large congenital are provided by the targeting into the
chromosomal duplications or deletions endogenous locus. Recently developed
in inborn genetic disease, specific dom- strategies take knockin significantly
inant mutations in oncogenes, recessive further and allow for specific intro-
mutations in tumorsuppressor genes, or duction of (point) mutations and re-
polymorphisms in genes involved in lipid peated exchange of genetic sequences:
metabolism linked to atherosclerosis. In recombination-mediated cassette ex-
order to understand the exact molecular change (RMCE) applies strategically
mechanism underlying the development positioned pairs of wt and mutated
of disease, such mutations need to be LoxP sites or LoxP and frt sites to
exactly recreated in the mouse. Clearly, achieve stepwise and directional re-
conventional and conditional genomic ma- placement of genomic sequences with
nipulations are not specific enough to 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- literature. Since the principal mecha-
tioning of LoxP sites on one chro- nism remains the same in all instances,
mosome allows for gene inversions a few of these applications are listed
or complete deletions, depending here, but not worked out in detail:
on the orientation of the recom- selection marker recycling, exchange of
binase recognition sites relative to reporter genes, removal of reporter genes,
each other (Fig. 14). Positioning of or selection markers (as in the generation
such recombinase recognition sites of floxed alleles).
on nonhomologous chromosomes has
proven to be a feasible strategy to
achieve chromosomal translocation,
5
alike translocations that are implicated Random Mutagenesis in ES Cells
in some leukemias.
– Targeted integration of transgenes to loci,
The completion of the mouse and human
which are known to be ‘‘open,’’ that
genome sequences has sparked renewed
is, not embedded in heterochromatin.
interest in developing tools for genome-
The gain of this approach is a high
wide mutagenesis in the mouse. Among
probability of transgene expression. An
often-used target locus is the ROSA26 the main molecular genetic technologies
locus, an ubiquitously transcribed lo- currently pursued are targeted mutagen-
cus that does not encode a protein, esis (see Sect. 3 and 4), insertional muta-
but which is active in many different genesis, by insertion of gene trap vectors
cell types throughout development and or viral or transposable sequences, and
adult life. Transgenes are flanked by chemical mutagenesis. These technologies
DNA sequences homologous to the all represent random mutagenesis proto-
ROSA26 locus and targeted via con- cols, which, in principle, can be applied
ventional methods (see Sect. 3). to mouse ES cells. The important advan-
– A substantial number of other appli- tage of random mutagenesis is that new
cations for site-directed recombination 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 Gene Trap Mutagenesis
identify and maintain recessive lethal phe-
notypes at any developmental stage, which, Gene trapping provides a method to
for instance, is not possible with chemical mutate genes in the genome through ran-
mutagenesis (see Sect. 5.3). 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 as Moloney Murine Leukemia Virus (Mo-
trap vectors insert a selectable marker MuLV) cause transformation by integra-
or reporter gene lacking essential regula- tion into the genome. Proviral integration
tory sequences into an endogenous gene. may activate oncogenes or interrupt tu-
Through marker insertion the gene is mor suppressor genes (Fig. 15b). This is
interrupted, while marker expression is an extremely useful asset in random mu-
complemented by endogenous regulatory tagenesis, since the integrated proviruses
elements (Fig. 15a). In this manner, tran- now serve as a molecular tag for cancer
scriptionally active as well as inactive loci genes: it can be used to identify the locus
can be trapped. Recent reports provided at which it is integrated by sequencing the
proof that gene trapping can also be used flanking integration site. Retroviral vectors
to selectively mutagenize genes that share have been adapted for use in mouse em-
certain properties, in this case, the fact that bryos and ES cells as well, and now permits
their gene products are all transmembrane germ-line mutagenesis.
proteins. A ‘‘secretory’’ trap vector was In analogy to random proviral insertion,
constructed such that it captured signal also transposable elements from yeast, in-
sequences in preceding exons by means of sects, fish, and mammals, among other
including a ‘‘capturing’’ transmembrane organisms, can be used for functional ge-
domain in the gene trap construct. These nomic analyses. Transposable elements
and other approaches collectively demon- (TEs) are a heterogeneous class of ge-
strate the power and versatility of gene netic elements that have the capacity to
trapping in genome-wide functional anal- move from one site on a chromosome to
ysis of molecular mechanisms important another: they ‘‘jump.’’ TEs are very effi-
in development and physiology. A col- cient at integrating into DNA, and some
lective gene trap database has recently elements can carry extra DNA. TEs are
been initiated by a number of consor- therefore useful vectors for transferring
tia (http://www.igtc.ca/index.html – http: new genetic material into genomes and
//www.escells.ca/ – http://www.mmrrc. for random insertional mutagenesis. By
org/ – http://tikus.gsf.de/project/web way of example, among the properties that
new/index.html – http://www.sanger.ac. make the Tc1/mariner superfamily of TEs
uk/PostGenomics/genetrap/ – http: useful for molecular genetic research are
//baygenomics.ucsf.edu/overview/ the facts that they are easy to work with,
welcome.html). their efficient and stable integration, and
persistent, long-term transgene expres-
sion following transposon-mediated gene
5.2 transfer. Importantly, such TEs ‘‘jump’’
Retroviruses and Transposable Elements in species other than their original hosts,
in Random Mutagenesis which underscores the need for fur-
ther development and use of these and
The use of retroviruses in random muta- alike molecular tools for functional ge-
genesis is adapted from applications in nomics in the mouse. Several reports on
which proviruses are used to screen for germline induced TE transposition and
collaborating oncogenes in leukemogene- germline attest to the mutagenic potential
sis. Slow transforming retroviruses such of the approach.
Transgenic Mice in Biomedical Research 539

5.3 variety of phenotypes, ranging from em-


Random Enu Mutation bryonic lethality to increased susceptibility
to chronic diseases such as cardiovascu-
For many years, mutant mouse models lar disease and cancer. The models allow
were obtained largely as a ‘‘spin-off’’ from defining primary gene functions as well
research on the mutagenic properties of as the role of a particular gene mutation
chemical compounds and radiation. With in disease.
the recent advent of more genome re- Once mouse models have been gener-
sources, it has become possible to identify ated, it is important to carry out an in-depth
such induced mutations more rapidly. Ini- analysis of the pathology of the entire
tially, substantial efforts were undertaken, mouse and not restrict the analysis to the
involving collaborations of many labora- target organs that are the main subject of
tories, each with their own phenotyping the intended study. A detailed strategy is
expertise. At present, robust technology outlined elsewhere. Often, one will find
is in place to mutagenize mice. One of that the dramatic alterations in the gene
the caveats remained however, because, al- expression pattern will show unexpected
though screens for recessive mutations are effects on the phenotype. For instance,
the most promising ones, such screens in the case of a study on apolipopro-
are exceedingly difficult and expensive. tein C1, the expected perturbation of lipid
Recently, it was shown that a narrowly metabolism was accompanied by a dra-
focused screening system for recessive matic and progressive hair loss of the mice
mutations can be highly productive, while with the highest level of expression of the
the expenses remained acceptable. More- transgene. The most common problem
over, M. Justice, A. Bradley, and colleagues encountered, however, in studies using ge-
made use of a set of strains carrying engi- netically modified mouse models is a lack
neered coat color markers and a chromo- of phenotype, even when a unique and
somal inversion (see Fig. 14a), enabling to evolutionarily conserved gene has been
further reduce costs by focusing the screen altered for which there are no obvious
on a predefined chromosomal segment. functionally redundant homologs present
Given the large number of loci controlling in the mouse genome. The main cause
complex genetic disorders, this strategy of this is the large difference in environ-
permits an ordered approach to finding mental stress experienced by wild mice as
disease genes. The strategy is highly suit- opposed to inbred laboratory mice. Often,
able to focus on some synthenic regions phenotypes need to be evoked by either
in the mouse genome, which are known increasing the environmental stress (i.e.
to be associated with disease susceptibility high-fat diets, mutagens) or increasing the
in man. genetic stress using a second transgenic
strain with a mutation in a related path-
way. A good example is provided by the
6 use of mouse strains in cardiovascular
Phenotype Analysis research. Wild-type laboratory mice are re-
sistant to develop atherosclerosis because
Genetically engineered mouse models of its highly antiatherogenic lipoprotein
serve to study genotype–phenotype rela- profile. By knocking out genes control-
tionships and can show an impressive ling lipoprotein metabolism, such as the
540 Transgenic Mice in Biomedical Research

apolipoprotein E gene, the mice become has been specifically modified to study its
sensitized. Upon feeding a moderated function. Another example is the study
high-fat diet, such mice will develop com- of cell migratory and (trans)differentiation
plex lesions in the aorta within 2 months. processes during neural tube closure; in
Many gene alterations will have no ef- these studies, one side of the tube is se-
fect on atherogenesis when studied sep- lectively electroporated with an expression
arately. However, when these mice are construct, while the other side functions
crossed with apolipoprotein E–deficient as a control.
mice, it is now possible to study the role The use of viral vectors to deliver trans-
of such genes. Thus, the apolipoprotein genes to mammalian cells is another
E–deficient mouse serves as a sensitized method to achieve partial transgenesis.
mouse model that is highly suscepti- The use of several classes and combina-
ble to the effects of additional genetic tions of viral vectors is being explored
changes. Likewise, changing the genetic in gene therapeutic applications. Similar
background of the mice will affect suscep- vectors are very useful in accomplishing
tibility to disease; this approach has been partial transgenesis, restricted to certain
used to define novel loci in human disease. tissues such as bone marrow (which is
Recent technological advances permit easily harvested and cultured ex vivo and
analysis of transgene expression and the reintroduced into a recipient mouse). The
biological consequences thereof by non- latter approach can also be employed to,
invasive methods. In vital imaging, very for instance, study isolated effects of gene
sensitive cameras measure biolumines- knockout in the hematopoietic compart-
cence from, for example, tumors or em- ment in combination with bone marrow
bryos in utero, which express luciferase of transplantation. Local and transient trans-
GFP reporter transgenes in living animals genesis is currently used to activate con-
(Fig. 2d). Clearly, the impact of technolog- ditional floxed transgenes (see Sect. 5), for
ical developments like these is substantial instance by adenovirus-encoded CRE. This
not only in terms of increased specificity approach was proven useful in, for ex-
and resolution of the model but also in ample, somatic activation of transforming
terms of reduced animal usage. oncogenes in the lung.
Lentiviral vectors, unlike the classical
slow replicating retroviruses, harbor the
7 advantage that they are not silenced in
Perspectives embryonic stem cells, which makes ex-
pression characteristics of such vectors
7.1 good. Recombinant lentiviruses were re-
Partial Transgenesis; Viral Shuttles cently used to introduce transgenes into
fertilized oocytes and ES cells (Fig. 6b).
Recent technological advances allow more The advantages of using lentiviral shuttle
rapid approaches to study gene function vector are significant: it obviates poten-
without the need for germ-line modifica- tially harmful procedures otherwise used
tion. Remarkably, partial transgenesis can to introduce transgenes into a cell (pronu-
be achieved through local electroporation clear microinjection, electroporation) and
of transgenic vectors into tissues. Using offers some degree of control over trans-
this approach, local muscle tissue in rats gene copy numbers.
Transgenic Mice in Biomedical Research 541

7.2 its subsequent enzymatic degradation. In-


RNA Interference; Posttranscriptional vestigations by many groups showed that
Gene Silencing RNA interference (RNAi) is a regulatory
process used by many organisms to con-
The abundance of biological information trol gene expression. Many of the protein
that has become available upon completion factors involved in RNAi are conserved,
of the genome sequence of both mouse and from protozoans to humans. RNAi has re-
human is exciting and overwhelming at cently been developed into a technology
the same time. An estimated 10 000 genes by which endogenous gene function can
have been identified currently, which rep- be assessed in many vertebrates, includ-
resents an estimated one-third of the total ing mammals. One obvious advantage of
gene complement in a human or mouse. RNAi-mediated gene silencing is that it
Of these genes, only a few thousand have does not rely on genetic modification. This
been functionally studied. Clearly, one of makes the technology very rapid compared
the remaining challenges in molecular ge- to (conditional) gene targeting. Proof of
netics is to analyze the function of novel principle has been achieved in many ver-
genes. Gene targeting is beyond any doubt tebrate systems including several human
one of the most successful and informative cells lines. RNAi also works in early mouse
technologies when it comes to elucidat- embryos, and was recently demonstrated
ing gene function. Despite improvements to be transmittable via the germline in
in cloning technologies that employ re- transgenic mice. Whereas initial strate-
combination in prokaryotes instead of gies in cell lines made use of synthetic
‘‘standard’’ recombinant DNA technology dsRNA sequences, a relative costly ap-
and that significantly simplify otherwise la- proach, RNAi, is currently achieved via
borious cloning strategies, gene targeting stable expression. In addition, recent re-
remains time-consuming and expensive. ports show that random siRNA libraries
Recent scientific and technological ad- can be generated from cDNA libraries via
vancements may, however, provide faster ingenious cloning strategies. The potential
means to address novel gene function. held by this approach is that such libraries
One of the most exciting developments are self-selecting, thereby enabling inves-
finds its origin in the realization that tigators to set-up high-throughput genetic
gene expression is controlled at a post- screens. RNAi can be made tissue specific,
transcriptional level by double-strand (ds) inducible, and can be delivered to mouse
RNA-mediated degradation of cytoplas- embryos using viral vectors. Such improve-
mic RNAs. This process, called RNA ments will clearly enhance the application
silencing – or posttranscriptional gene silenc- of RNAi in functional genomic research.
ing – was first discovered in plants as an Although genetic manipulation is possi-
unexpected gene silencing in transgenic ble in tissue culture, the study of genetic
plants: silencing of an endogenous gene interaction of transgenes with other genes
often occurred in concert with silencing of and local or systemic effects of transgene
the transgene. Transgene-mediated RNA expression in the intact organism provides
silencing is now recognized as a form of a much more complete and physiologi-
RNA silencing that uses small interfering cally relevant picture of a gene’s function
dsRNAs (siRNA), which serve as a guide than can ever be achieved in vitro. Ge-
for specific target mRNA recognition and netic modification therefore represents an
542 Transgenic Mice in Biomedical Research

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