Download Special Feature —Manipulating Genes to Understand

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Special Feature —Manipulating Genes
to Understand Cardiovascular Diseases:
Principles, Methodologies, and Applications
Major Approaches for Generating and
Analyzing Transgenic Mice
An Overview
Curt D. Sigmund
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Over the past decade, the development of gene-transfer technology in whole animals has afforded
unprecedented opportunities for investigators to probe complex regulatory systems in vivo. Important
advances in our understanding of the mechanisms of gene expression and regulation and the development
of animal models of human diseases are but two examples of how this technology has affected medical
science. Transgenic animals are defined as animals in which a segment of DNA has been physically
integrated into the genome of all cells, including the germ line, so that it can be transmitted to offspring
as a simple Mendelian trait The DNA segment generally consists of a whole cloned gene, cDNA, or a novel
gene modified by recombinant DNA methodologies. Whole genomic clones of genes are often used to study
tissue- and cell-specific expression and regulation or can be used to overexpress a gene product
Alternatively, the coding region of one gene can be fused to the transcriptional regulatory region of another
gene, causing it to be expressed in a new spectrum of tissues and cell types. A number of methods can be
used to introduce the DNA segment, including direct microinjection of one-cell fertilized embryos,
retroviral-mediated transfer, or gene transfer in embryonic stem cells. The technique most often used to
generate transgenic animals and perform "gene addition" experiments is direct microinjection. Alternatively, gene deletions or "knockouts" are performed by gene transfer in embryonic stem cells by
specifically targeting the site of integration in the genome. This methodology has the potential to be used
to generate specific gene mutations and has great applicability for the creation of animal models that
emulate human diseases (such as cystic fibrosis). An overview of the technology available for generating
transgenic animals, the relative merits of the different gene-transfer methods, and a brief description of
some of the questions that can be addressed using transgenic animals are discussed. (Hypertension.
1993^2:599-607.)
KEY WORDS
• mice, transgenic • transfection • genetics • renin • gene regulation
G
ene transfer (transfection) has become a routine
technique for delivering genetic material into
mammalian cells. The applications afforded by
this are too numerous to detail here, but they include
mapping of transcriptional regulatory elements and stimulating production of novel proteins for study. Although
transfection in vitro is an important tool and continues to
be extensively used, its applicability for probing the funcReceived March 16,1993; accepted in revised form June 1,1993.
From the Departments of Medicine and Physiology & Biophysics, University of Iowa College of Medicine, Iowa City.
Presented as a part of the Sunday Afternoon Program "Manipulating Genes to Understand Cardiovascular Diseases: Principles,
Methodologies, and Applications" at the American Heart Association's 65th Scientific Sessions, New Orleans, La, November 15,
1992.
This manuscript from the University of Iowa was sent to
Haralambos P. Gavras, MD, Consulting Editor, for review by
expert referees, for editorial decision, and for final disposition.
Correspondence to Curt D. Sigmund, PhD, Departments of
Medicine and Physiology & Biophysics, University of Iowa College
of Medicine, B150 Oakdale Research Park, 2501 Crosspark Rd,
Corarville, LA 52241.
tion played by specific genes in the pathogenesis of complex multigenic diseases such as hypertension, coronary
heart disease, and atherosclerosis is limited. Techniques
developed over the past decade to introduce genetic
material into the genome of whole animals have afforded
novel opportunities to examine the regulation of gene
expression and to correlate genotype with phenotype in
vivo. In this review I will discuss the methods used to
introduce genes into the mammalian genome and briefly
describe the types of experiments that can be performed
in transgenic animals. Where appropriate, specific terminology will also be defined. A number of general reviews
are available in the literature describing the use of transgenic animals in cardiovascular research,15 and some
specific examples of transgenic applications can be found
in the accompanying articles in this issue of Hypertension.
By definition, transgenic animals contain a segment of
exogenous DNA (a transgene) that has been physically
integrated into the genome of all cells, including the
germ line, and can be transmitted to offspring. A
number of methods have been described in the literature to accomplish this. Instead of providing an exhaus-
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FIG 1. Photomicrographs of a one-cell fertilized mouse embryo visualized with Nomarski
DIC optics. The egg is held in place by a holding
pipette (h), and the female (f) and male (m)
pronuclei are visible. The injection needle (i) is
resting outside the cell in A and has microinjected
a DNA solution into the male pronucleus of the
egg in B. Note the increase in pronuclear circumference in B. Equipment needed to perform
microinjections includes a microscope containing
a xlO eyepiece, x4 and x40 lenses, and
Nomarski DIC or Hoffman optics. Micromanipulators are mounted on each side of the microscope (one each for holding pipette and injection
needle) and can be hydraulic or mechanical
Microinjection can be controlled with a syringe
or pneumatic pump. Dissecting microscopes with
both direct and reflected illumination are needed
for egg manipulation and reimplantation; a microforge is needed to polish holding pipettes and
implantation pipettes; a pipette puller is needed to
reproducibly generate submicron tip injection
needles; and a humidity-controlled tissue culture
incubator is needed for culturing eggs.
tive review of these methods, I will briefly describe each
technique and their various benefits and pitfalls.
Generation of Transgenic Mice by Microinjection
By far the most common and well-characterized approach for producing transgenic mice is direct pronuclear microinjection of one-cell fertilized embryos. Microinjection is relatively easy to learn and perform, if
appropriate equipment is on hand (see legend to Fig 1),
and generally results in 5% to 40% of live births being
transgenic. Eggs obtained from superovulated female
donor mice are injected with an ultrathin drawn glass
capillary pipette (<1 nxn tip diameter) filled with a
solution containing the transgene construct of interest.
Approximately 1 to 2 pL of solution is delivered into the
male (larger) pronucleus until a visible increase in
pronuclear circumference is observed (Fig 1). The
surviving eggs are incubated until they divide to the
two-cell stage; then they are surgically reimplanted into
the oviducts of pseudopregnant foster mothers. Births, a
fraction of which are transgenic, occur 19 days later.
Female donor mice are hormonally superovulated by
the administration of pregnant mares' serum (PMS, 10
U IP) and human chorionic gonadotropin (hCG, 10 U
IP). The timing of these injections is fairly critical; the
PMS and hCG injections must be separated by 48 hours,
and the hCG injection must precede an endogenous
luteinizing hormone surge. The females are bred to
male mice overnight, and fertilization is confirmed by
the presence of a vaginal mucous plug in the morning.
The oviducts are surgically removed, and the eggs are
flushed into tissue culture media. A typical egg yield is
15 to 20 per oviduct; a variable fraction of which are
unfertilized, exhibit polyspermy, or are otherwise uninjectable. The pseudopregnant foster mothers are generated by breeding female mice with vasectomized or
genetically sterile males. A schematic representation of
the events described above is presented in Fig 2. Details
of these procedures, including a comprehensive review
of the equipment and protocols, have been published
previously.6-10
DNA segments delivered into the nucleus by microinjection integrate at random in the genome, and although multiple insertion sites have been reported, they
generally insert at a single site per diploid genome.
Finding multiple copies (in tandem head-to-tail arrays)
Sigmund
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of the transgene at the insertion site is a routine
occurrence, with the copy number ranging from one to
several hundred (Fig 3A). Unfortunately, transgene
expression is rarely proportional to copy number. In
addition, the random insertion of DNA can have several
detrimental consequences that need to be addressed in
a typical transgenic experiment. For instance, the tissue- and cell-specific expression profile exhibited by the
transgene can be altered as a result of juxtaposition of
Transgenic Mice: Methods and Analysis
601
FlG 2. Schematic shows time course of events during
production of transgenic mice. Injection of pregnant
mares' serum (PMS) is designated as day 1. Human
chorionic gonadotropin (hCG) is injected on day 3,
and matings between donor mice and male studs of the
same strain are set up overnight. On day 4, embryos are
removed from donors, briefly cultured, microinjected,
and cultured overnight until they divide to the two-cell
stage. Foster mothers are bred to vasectomized male
studs or genetically sterile males on day 4. Reimplantation of the two-cell stage-injected embryos is on day
5. Mice are born 19 days later (embryonic day 21, E21).
More details on the time course of this procedure have
been previously reported.6
the transgene near transcriptional regulatory elements
controlling another gene. The result, termed a "position
effect" or "position artifact," can be manifested by
higher or lower expression than normal, partially inappropriate, or completely inappropriate tissue-specific
expression.11 Examination of multiple independent
transgenic lines — each generated by an individual, and
therefore unique, insertion — allows investigators to distinguish these position artifacts from true expression.
B
11 Kb
B B
E
+
BamHI
B B
E
,
9 Kb
E
EcoRI
E
+
FIG 3. Identification of transgenic mice, with an example of Southern blotting (B), dot blotting (C), and potymerase chain
reaction (PCR) (D) shown for differentiating transgenic mice containing the human rerun gene from negative littermates. A:
Schematic map shows the organization of human renin transgenes in the genome. Multiple copies of the human renin gene are
arranged in a head-to-tail array. Two complete and two partial human renin gene units are shown for simplicity. The restriction
endonuclease sites used in the Southern blot are shown (B, BamHI; E, EcoRI). The position of the hybridization probe is indicated
by the closed bar; sizes of the fragments detected by the probe are indicated. B: Southern blot analysis of genomic DNA isolated
from tail biopsies and digested with BamHI (left) and EcoRI (right). In both blots the large molecular weight bands result from
fusion between the 3' end of one transgene unit and 5' end of the adjacent unit. The endogenous band in both blots is barely visible
at this exposure. C: Dot blot hybridization of undigested DNA loaded onto nitrocellulose using a manifold is hybridized with a
partial human renin cDNA probe. Identification of positive and negative transgenic mice is indicated. The copy number of the
transgene in this case easily allows us to differentiate transgene sequences from endogenous gene sequences. D: PCR analysis of
crude tail DNA preparations. PCR was performed using primers that specifically hybridize to the human renin gene and 35 rounds
of amplification. Identification of positive transgenic mice and negative littermates is indicated. The lower band in each lane is the
unextended oligonucleotide primers.
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Vol 22, No 4 October 1993
A second possible consequence of random integration is the potential to induce a mutation caused by the
disruption of an essential gene. Although these mutations can often go unnoticed until the animals are bred
to homozygosity (because only one chromosome of the
diploid set is affected), one needs to be aware of this
possibility when designing a breeding scheme for propagating a transgenic line. A variety of transgene-induced mutations have been reported in the literature. 1214 These mutations often have been extremely
interesting and have sparked further study (see below).
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Generating Transgenic Mice by Retroviral Transfer
and Embryonic Stem Cells
An alternative method for generating transgenic animals is infection of embryos with retroviral constructs.
A number of retroviral vectors are available that allow
genes to be inserted and packaged into viral particles.1516 These viral particles can be used to infect a
wide range of cell types, including embryos. Infection
can occur through direct exposure to retroviral particles
or by cocultivation with virus-producing cell lines. One
advantage of this technique is the ability to infect
preimplantation embryos at the 1-cell through the 8- or
16-cell stage. This has applicability to cell-lineage studies, which can benefit from the purposeful generation of
mosaic transgenic founder animals. Mosaic animals do
not contain a copy of the transgene in every cell, and
although mosaicism does occur in transgenic mice produced by microinjection, the process is unpredictable
and stochastic. However, it is important to realize that
the utility of the technique to study lineage relationships in mosaic animals is limited to the founder generation because all subsequent generations will contain
transgene DNA in all cells. An additional advantage of
retroviral-mediated transfer as compared with microinjection is the relative ease by which DNA sequences
flanking the position of the transgene insertion can be
cloned. The reason for this is that transgenes delivered
by retroviral infection integrate into the genome in a
single copy. The retrieval of flanking sequence, which
can be complicated by the presence of multimers of the
transgene in transgenics produced by microinjection, is
the first and most important step in identifying and
cloning genes mutated by transgene insertion. Disadvantages of the technique include the need to manipulate and produce viral particles and a limitation on the
size of the transgene genome being packaged (in the
8-kb range).
Both microinjection and retroviral-mediated gene
transfer suffer from the inability to specifically target
the chromosomal location of the insertion event. An
alternative to both of these methods is now gaining in
popularity because of the ability to precisely target
transgene insertion. This procedure involves the use of
embryonic stem (ES) cells and constructs designed to
homologoush/ recombine into the mammalian genome.
A review of these methods is described in an accompanying article in this issue of Hypertension.17 Briefly, ES
cells are plunpotent stem cells derived from the inner
cell mass of the mouse blastocyst. These cells can be
cultured under conditions that prevent their differentiation yet retain their potential to repopulate the entire
embryo. Furthermore, these cells can be genetically
manipulated by transfection or infection with constructs
designed to homologously recombine (exchange genetic
information) with regions of identical sequence on the
chromosome. The greatest advantage of this methodology is the ability to specifically mutate (create a null
mutation in) a gene located in the host genome. Because the cells are pluripotent, genetically modified ES
cells can be reintroduced into preimplantation mouse
blastocysts, where they become part of the inner cell
mass. These blastocysts can be reimplanted into the
uterus of a foster mother, where they will develop into
genetically mosaic animals. The mosaicism is due to a
mixture of parental blastocyst-derived and ES cellderived tissues. The extent of mosaicism can be easily
visualized if the ES cells and host blastocysts are derived
from mice with different coat colors. The resultant
mosaics have a mixture of colors, and the extent of
mosaicism is roughly reflected in the relative contribution of the two colors. Transmission of the disrupted
genes to offspring can occur if the germ line of the
mosaic animal is ES cell-derived, and germ line transmission of the ES cell genome results in mice with a
homogenous coat color, that is, ES cell-derived.
The greatest disadvantage of this technique is the
effort required to differentiate and detect a small number of homologous recombinants in a vast excess of
random integrants, although modifications in the design
of the recombination vectors can add an element of
selectivity to the process. For example, investigators
have had success using the herpes simplex virus thymidine kinase (HSV-TK) gene to enrich for specific targeting events. The HSV-TK gene, when expressed,
renders the cells sensitive to the drug gancyclovir, which
kills TK+ cells. If the gene is placed at either or both
ends of the targeting construct, it will remain intact and
integrate along with the transgene during random integration events but will be deleted if the transgene
integrates by homologous recombination. If a selectable
marker such as neomycin resistance is used as part of
the targeting construct, then selecting for neomycin and
gancyclovir resistance will enrich the overall population
(10- to 100-fold) for specific targeting events and therefore increase the chances of detecting the rare homologous recombinants.
Another common problem is the low frequency of
germ line transmission of the ES cell genome. Nevertheless, the potential to use the power of selective
mutation to the mammalian system outweighs such
technical difficulties. This is certainly borne out in the
recent development of important human disease models
in mice.18 An extensive review of factors influencing
homologous recombination in ES cells is available.1921
Analysis of Transgenic Animals:
Differentiating Transgenic Offspring From
Nontransgenic Littermates
In practice, transgenes integrate into the genome as
little as 5% or as much as 30% to 40% of the time.
Therefore, transgenic animals must be identified by
assaying for the presence of the transgene in the genome. Three general methods have been used to assay
for the presence of transgene sequences: restriction
endonuclease digestion of genomic DNA followed by
Southern blotting, dot blotting of undigested genomic
DNA, and the polymerase chain reaction (PCR) (examples of each are shown in Fig 3). Typically, the trans-
Sigmund Transgenic Mice: Methods and Analysis
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gene becomes part of the genome of all tissues, allowing
DNA to be purified from a small tail biopsy sample
without the need for an invasive surgical procedure.
Southern blotting, while being the most labor intensive
of the assays (because of the need for highly purified
DNA), is certainly the most stringent test for transgene
insertion. The most important use for Southern blotting
is when restriction fragment length polymorphism analysis is required to differentiate the transgene from a
closely related endogenous gene already present in the
mouse genome. We routinely perform Southern blotting
to identify putative transgenic founders.
Dot blotting (the application of denatured genomic
DNA directly to a solid membrane support) followed by
hybridization can be performed if the transgene sequences are unique to the mouse genome, such as a
bacterial reporter gene or viral oncogene. Homologous
sequences can be detected on dot blots if they are
present in sufficient copy. In the latter, the hybridization
intensity is used to differentiate transgenic from nontransgenic mice.
More recently, the application of PCR has been used
to rapidly identify transgenic animals. Because even
crude DNA preparations yield PCR amplification products, investigators are able to go from tissue sample to
results in a single day. The major requirement for
successful PCR is highly specific primer sequences that
are capable of differentiating transgene sequences from
endogenous gene sequences. Again, this is easy if the
transgene is unique to the genome. Minor sequence
polymorphisms, such as those present between different
alleles of a gene or interspecific genes, can easily be used
successfully to differentiate homologous sequences. In
this case, polymorphic residues are placed at the 3' end
of the oligomers, resulting in destabilization of the hybrid
formed between the oligonucleotide primer and a mismatched sequence. Application of PCR to identify transgenic mice containing the human renin gene is illustrated
in Fig 3.
Experimental Design: What Type of Construct
Should I Design?
Transgenic animal experiments are classically gene
addition experiments and can take several forms, including, among others, overexpression of protein products in normal and abnormal sites of synthesis, the use
of reporter genes for identifying cell types expressing a
gene, and mapping of transcriptional regulatory elements controlling gene expression. The design of the
transgene constructs outweighs all other considerations
in determining the types of questions that can be
addressed in transgenic animals. Fig 4 illustrates several
general classes of constructs that can be designed. The
remainder of this article will focus mainly on transgenic
mice generated by microinjection and gene addition
experiments.
Investigators have often been faced with the problem of
determining the function of a protein in regulating or
mediating a physiological response. One of the ways this
has been addressed is by studying the physiological consequences of acute or chronic protein overproduction in a
whole animal model. Indeed, this has been and continues
to be an important question in whole animal physiology.
Although short-term effects can be examined by direct
administration of a protein to an animal, elucidating
603
long-term effects can be much more laborious because of
difficulties in long-term delivery (days, weeks, or even
months) of a protein in vivo. Transgenic animals can be
designed to act as "chronic protein delivery systems"; in
essence, this is the quintessential question addressed in a
transgenic animal model. There are several approaches
through which this can be accomplished.
The first is overproducing a gene product in its
normal sites of synthesis. For this to be achieved, one
needs to be able to direct the synthesis of the gene
product to the appropriate cells, using transcriptional
regulatory elements active in that cell type and the
protein coding region of the gene being produced. For
all essential purposes, complete genomic clones that
contain all exons and introns and sequences extending
upstream (5') and downstream (3') of the gene already
have the information required to accomplish this.
Transgenic mice containing genomic clones that encode
the mouse and human renin genes have been successfully used to target expression to the appropriate spectrum of tissues and cells, with overexpression of the
mRNA and protein occurring in several tissues. 2327
Examples of human renin expression in transgenic
mice are shown in Fig 5 and also have been previously
reported.27 The data shown in the figure are from
transgenic mice containing a human renin genomic
segment including all 10 exons and introns and sequences extending approximately 900 bp upstream and
400 bp downstream of the gene. Fig 5A and 5B show
sample data on human renin transgene expression in the
adrenal gland of three different lines of transgenic mice.
Both mouse and human renin messages are visualized in
A, but only human renin mRNA is detected in B (see
legend). As can be seen, human renin mRNA is clearly
expressed in the adrenals of the transgenic animals, and
no human renin is observed in the adrenal glands from
nontransgenic DBA/2J (D) or C57BL/6 (B) mice. Adult
C57BL/6 mice do not express adrenal renin,23 and the
genetic background of the transgenics is based on a
strain with a similar adrenal renin expression profile.
Fig 5C shows a sample of human renin expression in the
kidney of a transgenic mouse and a nontransgenic
littermate. The assay is a differential primer extension
assay capable of differentiating the closely homologous
mouse and human renin mRNAs based on minor sequence polymorphisms.27 Approximately equal levels of
mouse and human renin mRNAs are expressed in
kidney. This is consistent with the observation that
approximately equal amounts of active mouse renin and
active human renin were released into the systemic
circulation (Fig 5D). These mice are currently being
used as models to examine the regulation of human
renin gene expression and secretion and also to determine if they will provide a model for examining the
regulation of human renin expression during experimental or genetic hypertension.
Clones comprising only a cDNA (DNA copy of an
mRNA) fused to regulatory sequences from a gene
(minigenes) can also be used to target cell-specific
expression, although the use of cDNA clones has been
less predictable than the use of genomic clones. This is
apparently due to the observation that the presence of
spliceable intron sequences is required for high-level
expression and proper maturation of an mRNA. The
use of a DNA cassette containing a cloned heterologous
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Hypertension
Construct Type
Genomic
"mini-gene"
Vol 22, No 4
October 1993
Explanation
General Structure
DNA segment from
genomic clone containing
5' and 3' flank, all exons
and introns
DNA segment containing 5'
flanking sequence fused to
cloned cDNA and artificial
intron + poly A site.
13' tunic
L- Inuonj
'—I
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Reporter Fusion
Oncogene Fusion
DNA segment containing
promoter element from
another gene fused to the
protein coding region of
interest
DNA segment containing 5'
flanking region of interest
fused to reporter gene.
DNA segment containing 5'
flanking region of interest
fused to oncogene
Study tissue- and cell-specific expression
Overproduce gene product in normal sites
of synthesis. Transgenes should respond
to signals which normally regulate
expression of gene
Essentially the same as for genomic
constructs Constructs are generally
expressed better if artificial intron and poly
A addition sites are added Most important
when genomic clone spans large distance.
5' flank I
Heterologous exons
Promoter
^ ' '±
HeterologousPromoter
Function
poiv A
used to impart a new tissue- and cellspecific expression profile on the gene
being expressed. By using a well
characterized heterologous promoter a
3- fi»k 9 e n e ' s mRNA and product can be redirected to a new spectrum of tissues and
cells.
^
Hatarologoui
Promoter
5' fiani< reporter gene
lacZ. CAT.
luciferase
DQ|V A
intron
5' f l a n k y i n t r o n M a ^ ^ a w
oncogene such as
SV40 Tag, ras, etc.
Used to examine the cellular sites of gene
expression and mapping transcriptional
regulatory elements in the 5' flanking region
of a gene. Expression of bacterial genes
may benefit from intron and poly A site.
Similar to reporter gene in that promoter
element directs synthesis of oncogenic
product to specific cells. Oncogene can be
used as reporter. Most often used to target
dysplastic or neoplastic growth to specific
cells and for isolating novel cell lines
FIG 4. Summary of the various types of constructs that can be designed for use in transgenic mice. A schematic representation
of each construct is included, in which the hypothetical gene of interest has four exons (horizontal hatching) separated by introns
in the genomic context but spliced together in the minigene context. The promoter for this gene is indicated by a thin line labeled
5' flank. A heterologous promoter is so labeled and indicated by a thick line. The intron-poty A segment used in the minigene,
heterologous promoter, and reporter constructs can be any of a number of DNA cassettes containing a cloned intron and
polyadenylation recognition site. The various DNA segments are assembled using standard molecular cloning techniques.22 CAT,
chloramphenicol acetyltransferase.
intron linked to a polyadenylation recognition site may
help eliminate such problems (Fig 4). Expression of a
mouse angiotensinogen minigene has been previously
reported in transgenic mice.28
A limiting factor in overproduction of a gene product
using its endogenous promoter elements may be the
tight restriction imposed on the level of gene transcription, which is caused by the presence of regulatory
signals in the promoter designed to prevent uncontrolled overproduction of the final protein product. For
example, overproduction of renin in the kidney can
potentially induce a cascade of events whereby elevated
levels of circulating angiotensin II feed back on the
kidney and attenuate expression of the renin gene.29
Regulatory input may also be received as a result of
physiological perturbation. Using the same example,
increased arterial pressure caused by elevated circulating angiotensin II could trigger baroreceptors and cause
a reduction in expression of the renin gene in the
kidney. Transgenic rats containing a mouse renin genomic construct exhibit a markedly elevated arterial
pressure and severely attenuated level of renal renin
mRNA.30
An alternative method for overproducing a protein is
to place the protein coding region of the gene under the
control of a heterologous promoter element that is
unresponsive to regulatory inputs controlling expression
of the gene. Overproduction of atrial natriuretic factor
in the liver of transgenic mice was achieved by fusing the
coding region of atrial natriuretic factor to the liverspecific transthyretin promoter.31'32 The results were
constitutive overproduction of atrial natriuretic factor in
liver, elevated circulating levels of the hormone, and
chronically lowered arterial pressure. Similarly, expression of the rat renin and rat angiotensinogen genes
under the control of the metallothionein promoter
resulted in elevated expression of both genes in liver,
elevated levels of both proteins in plasma, and mild but
chronic hypertension.33 Currently, the limiting factor in
directing the synthesis of any protein to any cell type is
the availability of well-characterized, cell-specific regulatory elements (promoters). Certainly, as additional
genes are identified and cloned, our ability to target
specific cell types will improve.
Of course, of equal or potentially greater import is
protein underproduction. ES cell technology, discussed
Sigmund
Transgenic Mice: Methods and Analysis
605
D
HuRen Tg+
D B
B
200
Tg+ Tg-
it
-Mouse
-Human -jj E
Total
Mouse
Human
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FIG 5. Examples of tissue-specific expression analysis of transgenic mice containing a human renin genomic clone are shown.27
A and B: Northern blot of adrenal gland RNAs from various adult transgenic mice (Tg+) and nontransgenic DBA/2J (D) and
BCF (B) mice. The blot in A is probed under conditions that allow us to detect both human renin and endogenous mouse renin
mRNA. BCF mice do not express renin in the adrenal gland at this age. The genetic background of the transgenic mice is BCF.
The blot in B is probed under conditions that allow us to detect only human renin mRNA. See Reference 27 for further details.
C: Differential primer extension analysis of kidney RNA from a transgenic (Tg+) mouse and nontransgenic (Tg-) littermate. The
assay is capable of generating specific bands correlating to the expression of the human and mouse renin gene. No human renin
mRNA is detected in the kidney of the nontransgenic animal. Approximately equal levels of human and mouse renin mRNA are
expressed in the transgenic kidney. D: Bar graph shows plasma renin activity assay. Plasma was isolated from transgenic and
nontransgenic animals and subjected to a plasma renin activity assay using a substrate cleavable by both human and mouse active
renin. Total: Combined human and mouse active renin activity in transgenic mice. Mouse: Level of mouse active renin activity in
transgenic and nontransgenic mice. Human: Level of active human renin activity in transgenic mice. The level of human and
mouse renin was extrapolated by subtracting the plasma renin activity of nontransgenic mice (data not shown). We previously
demonstrated that the presence of the human renin transgene has no effect on the expression or release of active mouse renin.27
above, while having the ability to knock out expression
of a gene in all tissues, is not tissue selective. A
"tissue-specific knockout" utilizing a targeted antisense
RNA has been performed successfully in transgenic
mice.34 Antisense RNA is generated by placing the
coding region of a gene in the opposite orientation with
respect to a cell-specific promoter. Theoretically, the
antisense RNA hybridizes to the normal mRNA and
either renders it translationally inactive or targets the
duplex for degradation; if targeted to a specific cell type,
this could address important questions regarding the
function of a gene product in a single tissue or set of
tissues. In practice, however, such experiments have
proved to be extremely difficult. Factors influencing the
success of an antisense experiment probably include the
availability of a powerful cell-specific promoter, the
level of antisense RNA production (needed in extreme
molar excess), and the stability of the antisense mRNA.
Also, in the case of an inhibition of translation, the
antisense mRNA has to traverse the nucleus and accumulate in the cytoplasm where protein synthesis occurs.
Perhaps the development of improved tools, such as
ribozymes (catalytic antisense RNAs), will eventually
make these experiments more feasible.
Our ability to express mRNAs in specific cell types in
transgenic animals has allowed us to specifically target
proteins, such as bacterial genes and viral oncogenes,
that are not normally expressed in mammals. This has
been an extremely powerful technique, providing us
with an unprecedented opportunity to study gene expression in vivo, identify expressing cell types, develop
novel cell lines, and emulate human disease. Reporter
genes generally encode innocuous gene products that
can easily be detected through a biologic or histochemical assay and have been extensively used in tissue
culture for many years. The most popular reporter gene
in transgenic mice has been E. coli lacZ encoding
/J-galactosidase. /3-Galactosidase expression can be detected easily in tissues by virtue of its unique mRNA but
more importantly by its catalytic activity. A number of
synthetic substrates are available, which when cleaved
by /3-galactosidase, liberate color, making it an extremely sensitive reporter. A number of questions can
be addressed when /3-galactosidase is expressed in
transgenic mice using cell-specific promoter elements:
(1) In what cell types is a promoter active? (2) Does the
cell specificity of the promoter change throughout ontogeny? (3) How is the gene regulated? Transcriptional
regulatory elements can also be mapped using this
approach by analyzing a collection of /3-galactosidase
constructs differing in the amount of 5' flanking sequences used to target expression. Examples of transgenic mice containing the y3-galactosidase gene have
been previously reported35-36; other reporter genes, such
as chloramphenicol acetyltransferase and luciferase,
among others, have also been used successfully.37-38
Some oncogenes, by virtue of their uniqueness in the
mammalian genome, also can be used as a reporter.
Transgenic mice containing the mouse renin promoter
fused to SV40 T antigen were previously used to map
regulatory elements in the renin gene.39-40 More significant, however, is the ability to target an oncogene to
specific cells, causing, in many cases, a dysplastic or
neoplastic phenotype to result. These types of studies
have been instrumental in examining the mechanisms of
tumorigenesis and developing animal models for the
study of tumor progression.4143
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October 1993
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The accumulation of an oncoprotein in a cell can
predispose it to a series of genetic modifications, leading
to a fully tumorigenic phenotype. These cells, once
amplified in a tumor, are often immortalized or fully
transformed and therefore can be propagated permanently in vitro. The renin promoter SV40 T antigen
transgenic mice alluded to above have been used to
isolate a renin-expressing kidney cell line that exhibits
many characteristics of juxtaglomerular cells.44-46 Similarly, atrial tumors and atrial cardiomyocyte lines have
been established from transgenic mice containing the
ANF promoter fused to T antigen.47 A concern in such
experiments is that the transformation event leading to
the immortalized phenotype can affect the terminaldifferentiated character of the cells. An important question that must be addressed in these experiments is how
the resultant cell lines emulate their in vivo counterparts. Recently, conditionally active oncogenes, such as
temperature-sensitive mutants of SV40 T antigen, have
been used to limit the effects of the transformation
process.48
Conclusion
Transgenic animals have provided and continue to
provide an important resource for cardiovascular research. Novel animal models exhibiting hypertension
and atherosclerosis are providing important new data
on the pathogenesis of these diseases and the role
played by specific genes. Recently, the power of molecular genetics has been used to identify genes linked to
hypertension. Transgenic animals will unquestionably
become an important tool for testing the significance of
allelic gene variants. Combined with the ability to
specifically mutate genes involved in cardiovascular
regulation, the use of transgenic animals will undoubtably lead to our greater understanding of genotype and
phenotype in the cardiovascular system.
Acknowledgments
Work presented herein was supported by grants HL-48058
and HL-48459 from the National Institutes of Health, Bethesda, Md, and grants from the American Heart Association
and the Carver and Lindsay Trusts at the University of Iowa.
C.D.S. is an Established Investigator of the American Heart
Association. I wish to thank Julie A. Lang and Norma L.
Sinclair for their excellent technical assistance with several of
the studies presented in this review. I also wish to thank W.
Michael Sinclair and John M. Burson for their critical review
of the manuscript.
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Hypertension. 1993;22:599-607
doi: 10.1161/01.HYP.22.4.599
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