21.2.1 Transgenic animals can be produced following transfer of cloned DNA into fertilized oocytes and cells from very early stage embryos

Transgenesis involves transfer of foreign DNA into totipotent or pluripotent embryo cells (either fertilized oocytes, cells of the very early embryo or cultured embryonic stem cells) followed by insertion of the transferred DNA into host chromosomes. If the foreign DNA integrates into the chromosomes of a fertilized oocyte, the developing animal will be fully transgenic since all nucleated cells in the animal should contain the transgene. If chromosomal integration occurs later, at a postzygotic stage, the animal will be a mosaic, with some cells containing the transgene and some others lacking it. If the transgene is present in germline cells it can be passed through sperm or egg cells into some of the animal's progeny, and PCR-based tests can be used to quickly screen for the presence of the transgene. Progeny that are transgene positive can be expected to be fully transgenic; all their cells should have developed from a fertilized oocyte containing the transgene.

Pronuclear microinjection

To obtain transgenic mice by this route, females are superovulated, mated to fertile males and sacrificed the next day. Fertilized oocytes are recovered from excised oviducts. The DNA of interest is then microinjected using a micromanipulator into the male pronucleus of individual oocytes (Figure 21.1). Surviving oocytes are reimplanted into the oviducts of foster females and allowed to develop into mature animals (see Gordon, 1992).

Figure 21.1

Construction of transgenic mice by pronuclear microinjection. Very fine glass pipettes are constructed using specialized equipment: one, a holding pipette, has a bore which can accommodate part of a fertilized oocyte, and thereby hold it in place, while (more...)

During this procedure, the microinjected DNA (transgene) randomly integrates into chromosomal DNA, usually at a single site, although rarely two sites of integration are found in a single animal. Individual insertion sites typically contain multiple copies of the transgenes integrated into chromosomal DNA as head-to-tail concatemers (it is not unusual to find 50 or more copies at a single insertion site). As a result of chromosomal integration, the transgenes can be passed on to subsequent generations in mendelian fashion: if the foreign DNA has integrated at the one-cell stage, it should be transmitted to 50% of the offspring.

21.2.2 Cultured embryonic stem (ES) cells provide a powerful route to genetic modification of the germline

The microinjection of foreign DNA into fertilized oocytes is technically difficult and not suited to large-scale production of transgenic animals or to sophisticated genetic manipulation. A popular alternative, but one which has so far been restricted to the construction of genetically modified mice, involves transferring the foreign DNA initially into cultured embryonic stem (ES) cells. Mouse ES cells are derived from 3.5–4.5 day postcoitum embryos and arise from the inner cell mass of the blastocyst (see Figure 21.2). The ES cells can be cultured in vitro and retain the potential to contribute extensively to all of the tissues of a mouse, including the germline, when injected back into a host blastocyst and reimplanted in a pseudopregnant mouse.

Figure 21.2

Genetically modified ES cells as a route for transferring foreign DNA or specific mutations into the mouse germline. Cells from the inner cell mass were cultured following excision of oviducts and isolation of blastocysts from a suitable mouse strain (more...)

The developing embryo is a chimera: it contains two populations of cells derived from different zygotes, those of the blastocyst and the implanted ES cells. If the two strains of cells are derived from mice with different coat colors, chimeric offspring can easily be identified (see Figure 21.2). Use of genetically modified ES cells results in a partially transgenic mouse. Because the injected ES cells can form all or part of the functional germ cells of the chimera, it is possible to derive fully transgenic mice. This is usually accomplished by screening the offspring of matings between chimeras (usually males) and mice with a coat color recessive to that of the strain from which the ES cells were derived (see Figure 21.2).

The big advantage of ES cells is that they can be grown readily in culture. This means that a variety of genetic manipulations can be conducted in cultured ES cells. Importantly, the desired genetic modification can be verified in tissue culture, before injecting the genetically modified cells into a blastocyst prior to implantation. For example, the desired gene can be ligated to a marker gene, such as the neo gene, enabling a positive selection for cells that have been successfully transfected (see Box 10.1). The presence of the desired gene can also be verified quickly by a PCR-based assay. ES cells also offer the huge advantage of gene targeting by homologous recombination, a method which can permit a programed selective alteration of a single predetermined gene and also highly specific ways of chromosome engineering. Such approaches are extremely powerful for understanding gene function (Section 21.3) and also for creating animal models of disease (Section 21.4).

The ES cell approach to constructing transgenic mice was made possible by the successful establishment in the early 1980s of stable cell lines from isolated mouse ES cells. ES cells were not so readily identifed in other mammals, although there have been some important recent successes (see Box 21.1).

Box 21.1

Isolation and manipulation of mammalian embryonic stem cells. The embryo proper derives from the inner cell mass (ICM) of a blastocyst (Box 2.2). Mouse embryonic stem (ES) cells were first isolated from blastocysts by Evans and Kaufman (1981) and Martin (more...)

21.2.3 Transgenic animals have been used for a variety of studies investigating mammalian gene expression and function

Transgenic animals have been extremely important for analyzing human genes (Hanahan, 1989; Camper et al., 1995; Theuring, 1995), and have helped greatly in our understanding of a variety of fundamental biological processes, notably in immunology, neurobiology, cancer and developmental studies. The following list is far from exhaustive but is intended to illustrate some major types of application:

• Investigating gene expression and its regulation. Although evidence for cis-acting regulatory elements is often inferred initially from studies using cultured cells, they need to be validated in whole animal studies. Transgenes consisting of the presumptive regulatory sequence(s) coupled to a reporter gene, such as lacZ, provide a sensitive method of detecting gene expression and a powerful way of investigating regulation of gene expression. Long-range control of gene expression is often investigated using YAC transgenes, see Section 21.2.4.
• Investigating gene function by targeted gene inactivation. Specific genes can be inactivated by a gene targeting procedure to introduce a transgene into the target gene (insertional inactivation). The effect on the phenotype of creating a null mutation in the gene of interest can provide powerful clues to gene function.
• Investigating dosage effects and ectopic expression. In some cases, valuable information can be gained by over-expressing a transgene (e.g. Schedl et al., 1996) or by expressing it ectopically (the transgene is coupled to a tissue-specific promoter which causes expression in cells; the phenotypic consequence may provide valuable clues to function).
• Cell lineage ablation. Transgenes can be designed consisting of a tissue-specific promoter coupled to a sequence encoding a toxin, for example diphtheria toxin subunit A or ricin. When the promoter becomes active at the appropriate stage of tissue differentiation, the toxin is produced and kills the cells. Thus, certain cell lineages in the animal can be eliminated (cell ablation) and the phenotypic consequences monitored.
• Investigating gain of function. In principle, any mammalian gene that produces a dominant negative effect or gain of function can be investigated by introduction of an appropriate transgene. In some cases, this can provide proof of a suspected biological function. A classical example concerns the Sry gene. A variety of different genetic analyses had implicated this gene as a major male-determining gene but convincing proof was obtained using a transgenic mouse approach. The experiment consisted of transferring a cloned Sry gene into a fertilized 46,XX mouse oocyte. As a result of this artificial intervention, the resulting mouse, which nature had intended to be female, turned out to be male (Koopman et al., 1991).
• Modeling human disease. Insertional inactivation is often used to model loss of function mutations whilst gain of function mutations can often be modeled by inserting a mutant transgene (see Section 21.4).

21.2.4 The use of YAC transgenes and inducible promoters has greatly extended the applications of transgenic animals

YAC transgenics

Early studies of gene expression and regulation in transgenic animals involved transfer of small genes. However, expression of small transgenes often fails to follow the normal temporal and spatial patterns of expression or match the expression level of the endogenous homolog. An increasing number of human genes are known to be very large (see Figure 7.7). Even in the case of small genes, important regulatory elements that are required for correct expression may be located many kilobases upstream of the coding sequence (see Figure 8.23). In order to be able to study the expression and regulation of a human gene under the control of its own cis-acting regulatory elements, it was therefore necessary to establish transfection conditions which would allow the transfer of large DNA clones.

A major breakthrough in transgenic studies was the development of so-called YAC transgenics (Lamb and Gearhart, 1995). The first report to be published described transfer of a 670 kb YAC containing the human HPRT (hypoxanthine phosphoribosyltransferase) gene into mouse ES cells (Jakobovits et al., 1993). This was accomplished by spheroplast fusion (i.e. fusion of ES cells with YAC-containing yeast cells that have been stripped of the hard cell wall; see Section 4.3.4). Fragments from the yeast genome can integrate at the same time, however, and so alternative methods have sought to purify an individual YAC by size-fractionation on a preparative gel using pulsed-field gel electrophoresis (assuming that the YAC migrates at a position in the gel that is different from any yeast chromosome). The purified YAC can be inserted into a fertilized oocyte by pronuclear microinjection (see above). This method is, however, limited to small YACs: the DNA of large YACs is more likely to fragment following microinjection with very fine micropipettes. Alternatively, purified YACs have been transferred into ES cells by using liposomes, artificial lipid vesicles that are used to transport molecules into a cell following fusion of the lipid coat with the plasma membrane of the recipient cell (see Figure 22.6).

YAC transgenics have permitted study of large genes such as the 400 kb human APP gene, a gene known to contribute to Alzheimer's disease, encoding the amyloid protein precursor. A YAC transgene containing APP showed tissue- and cell type-specific expression patterns closely mirroring that of the endogenous mouse gene (for this and other examples, see Lamb and Gearhart, 1995). Long-range gene regulation mechanisms (locus control regions, imprinting and other chromatin domain effects; see Chapter 8) can be modeled. An interesting application has been in the production of fully human antibodies in the mouse by transfer of human YACs containing large segments of the human heavy and kappa light chain immunoglobulin loci into mouse ES cells, and thence the creation of transgenic mice able to produce human antibodies (see Mendez et al., 1997 and Figure 21.3). Finally, YAC transgenics may also find a role in modeling disease caused by large-scale gene dosage imbalance (see Section 21.4.4).

Figure 21.3

Use of YAC transgenesis to construct a mouse with a human antibody repertoire. YACs containing Ig sequences are obtained by screening YAC libraries with suitable Ig probes. The recovery of YACs with comparatively small inserts meant that there was a need (more...)

21.3.1 Gene targeting by homologous recombination in ES cells can be used to produce mice with a mutation in a predetermined gene

Gene targeting involves engineering a mutation in a preselected gene within an intact cell. It can therefore be viewed as a form of artificial site-directed in vivo mutagenesis (as opposed to the various methods of site-directed in vitro mutagenesis described in Section 6.4). The mutation may result in inactivation of gene expression (a ‘knock-out’ mutation), or altered gene expression, and so can be useful for studying gene function (see below). In addition, the same method can be used to ‘correct’ a pathogenic mutation by restoring the normal phenotype, and so has therapeutic potential (see Section 22.3).

Gene targeting typically involves introducing a mutation by homologous recombination. A cloned gene (or gene segment) closely related in sequence to endogenous target gene is transfected into the appropriate cells. In some of the cells, homologous recombination occurs between the introduced gene and its chromosomal homolog. Gene targeting by homologous recombination has been achieved in some somatic mammalian cells, such as myoblasts. However, the most important application involves mouse ES cells: once a mutation has been engineered into a specific mouse gene within the ES cells, the modified ES cells can then be injected into the blastocyst of a foster mother and eventually a mouse can be produced with the mutation in the desired gene in all nucleated cells (Capecchi, 1989; Melton, 1994).

Homologous recombination in mammalian cells is a very rare occurrence (unlike in yeast cells, for example, where it occurs naturally at high frequencies, enabling sophisticated genetic manipulation). The frequency of homologous recombination is increased, however, when the degree of sequence homology between the introduced DNA and the target gene is very high. As a result, the introduced DNA clone is a mouse sequence which should preferably be isogenic (derived from the same mouse strain as the strain of mouse from which the ES cells were derived). Even then, the frequency of genuine homologous recombination events is very low and may be difficult to identify against a sizeable background of random integration events.

To assist identification of the desired homologous recombination events, the targeting vector (often a plasmid vector) contains a marker gene, such as the neo gene (see Box 10.1), which permits selection for cells that have taken up the introduced DNA. PCR assays are used to screen for evidence of a homologous recombination event (by using a marker-specific primer plus a primer derived from a sequence present in the target gene but absent from the introduced homologous gene segment). The targeting construct is transferred into cultured mouse ES cells by electroporation, a method in which pulses of high voltage are delivered to cells, causing temporary relaxation of the selective permeability properties of the plasma membranes. Two basic approaches have been used:

• Insertion vectors target the locus of interest by a single reciprocal recombination, causing insertion of the entire introduced DNA including the vector sequence (Figure 21.4A). This is the most reliable way of causing a knock-out mutation.
• Replacement vectors are designed to replace some of the sequence in the chromosomal gene by a homologous sequence from the introduced DNA (Figure 21.4B). This can occur as a result of a double reciprocal recombination or by gene conversion. The replacement method can inactivate a gene when the introduced sequence contains one or more premature termination codons or lacks critical coding sequences. It can also be used to correct a pathogenic mutation.

Figure 21.4

Gene targeting by homologous recombination can inactivate a predetermined chromosomal gene within an intact cell. (A) Insertion vector method. The introduced vector DNA (blue) is cut at a unique site within a sequence which is identical or closely related (more...)

The replacement vector approach, as well as the insertion vector method, often leaves foreign sequences at the target locus. In some cases, however, a more subtle mutation is required. For example, it may be desirable to investigate the effect of changing a single codon. Various two-step recombination techniques can be used to accomplish this method, and the resulting mouse, although genetically modified lacks any foreign sequences and so can no longer be described as transgenic (see Figure 21.5 and Melton, 1994).

Figure 21.5

Double replacement gene targeting can be used to introduce subtle mutations. Both the methods in Figure 21.4 result in introduction of a substantial amount of exogenous sequence within the endogenous gene. To introduce a subtle mutation without leaving (more...)

Gene targeting in mice is popularly used for producing artificial mouse models of human disease (Section 21.4). In addition, it provides a powerful general method of studying gene function. The gene in question is selectively inactivated, producing a ‘knock-out’ mouse, and the effect of the mutation on the development of the mouse is monitored carefully. Sometimes there is little or no phenotypic consequence after inactivating a gene that would be expected to be crucially important, such as some genes which encode a transcription factor known to be expressed in early embryonic development. The lack of a phenotype in such cases is often thought to be due to genetic redundancy (another gene is able to carry out the function of the gene that has been knocked out). As a result, in some cases double or even triple gene knock-outs have been carried out to analyze gene function, as in the case of some of the Hox genes (see, for example, Manley and Capecchi, 1997).

A useful example of investigating functional redundancy concerns studies of the mouse Engrailed genes, En-1 and En-2. Both of these genes are homeobox genes which had been considered to play crucial roles in brain formation. En-1 knock-outs have serious abnormalities but surprisingly En-2 knock-outs have only minor problems. Expression of the En-1 gene is switched on 8–10 hours before that of the En-2 product, suggesting that perhaps the En-1 product can compensate for the lack of En-2 product in En-2 knock-outs. To test for the possibility of functional redundancy, Hanks et al. 1995 used a variant of the knock-out procedure known as the ‘knock-in’ technique. In this case the transgene used to knock-out the target endogenous gene is itself designed to be expressed under the control of the cis-acting elements of the knocked-out gene. A transgene containing an En-2 coding sequence was used to knock-out the endogenous En-1 gene. In so doing, the introduced En-2 sequence came under control of the En-1 regulatory sequences and was expressed before the endogenous En-2 gene was switched on (see Figure 21.6). The resulting En-1 knock-out mouse had a normal phenotype, demonstrating that the knocked-in En-2 gene was functionally equivalent to En-1 (Hanks et al., 1995).

Figure 21.6

The knock-in method replaces the activity of one chromosomal gene by that of an introduced gene. The En-1 gene shown at top has two exons and coding sequences are shown by filled boxes. Its promoter (P) and polyadenylation site (pA) are also shown. The (more...)

21.3.2 Site-specific recombination systems, notably the Cre-loxP system, extend the power of gene targeting

Several site-specific recombination systems from bacteriophages and yeasts have been characterized and are promising tools for genome engineering (Kilby et al., 1993). Thus far, the Cre-loxP recombination system from bacteriophage P1 has been the most widely used. The natural function of the Cre (causes recombination) recombinase is to mediate recombination between two loxP sequences that are in the same orientation (the loxP sequence consists of 34 bp and comprises two inverted 13 bp repeats separated by a central asymmetric 8 bp spacer; Figure 21.7). As a result of recombination, the intervening sequence between the two loxP sites is excised (see Figure 4.15). Using gene targeting, loxP sequences can be stitched into a desired gene or chromosomal location, and the subsequent provision of a gene encoding the Cre product can result in an artificially generated site-directed recombination event (see Chambers, 1994). As described below, several applications can be envisaged (Lobe and Nagy, 1998).

Figure 21.7

Structure of the loxP recognition sequence. Note that the central 8 bp sequence which is flanked by the 13 bp inverted repeats is asymmetric and confers orientation.

Tissue- and cell type-specific knock-outs

Some genes are vital to early development and simple knock-out experiments are generally not helpful because death ensues at the early embryonic stage. To overcome this problem, methods have been developed to inactivate expression of the target gene in only selected, predetermined cells of the animal (conditional knock-out). The animal can therefore survive and the effect of the knock-out can be studied in a tissue or cell type of interest.

An early example of a conditional knock-out sought to study the role of DNA polymerase β (an enzyme that is essential for embryonic development) in T lymphocytes (Gu et al., 1994). The gene targeting procedure replaced part of the endogenous gene by an introduced homologous gene segment flanked by loxP sequences. Mice carrying this targeted mutation were then mated with a strain of mice which carried a Cre transgene gene under the control of a T cell-specific promoter. Offspring with the loxP-flanked pol β sequences plus the Cre transgene were identified and survived to adulthood. The Cre product was expressed only in T cells, leading to inactivation of the target gene in these cells by excision of DNA polymerase β gene segment between the two loxP sequences (see Figure 21.8 for the general method).

Figure 21.8

Gene targeting using the Cre-loxP recombination system can be used to inactivate a gene in a desired cell type. (A) Illustration of a standard homologous recombination method using mouse ES cells, in which three loxP sites are introduced along with a (more...)

Chromosome engineering

Another important recent development is a strategy for chromosome engineering in ES cells which relies on sequential gene targeting and Cre-loxP recombination. Gene targeting is used to integrate loxP sites at the desired chromosomal locations and, subsequently, transient expression of Cre recombinase is used to mediate a selected chromosomal rearrangement (Ramirez-Solis et al., 1995; Smith et al., 1995; Figure 21.9). Chromosome engineering strategies of this type offer the exciting possibility of creating novel mouse lines with specific chromosomal abnormalities for genetic studies.

Figure 21.9

Chromosome engineering can be accomplished using cre-loxP systems. (A) Use of targeted insertion of loxP sites to facilitate a chromosomal translocation. See Smith et al. 1995 for a practical example. (B) Use of targeted insertion of loxP sites to permit (more...)

The multiple targeting and selection steps in ES cells used in the above chromosome engineering methods can be avoided using the novel approach of Herault et al. 1998. This targeted meiotic recombination method takes advantage of the homologous chromosome pairing that occurs naturally during meiosis at the first cell division. A transgene is designed to express Cre recombinase under the control of a Sycp1 promoter (the Sycp1 gene encodes the SCYP1 protein which is part of the synaptonemal complex that facilitates crossing over). As a result Cre recombinase is produced in male spermatocytes during the zygotene to pachytene stages when chromosome pairing occurs.

21.3.3 Gene trapping in mouse ES cells allows an efficient approach to functional analysis

Large-scale approaches to investigating animal gene function have principally relied on exposing animals to high doses of radiation, or to potent chemical mutagens such as ethylnitrosurea (ENU) or ethyl methylsulfonate (EMS). In mouse mutagenesis programs, for example, males are typically exposed to high levels of a suitable mutagen to induce a high frequency of mutation in sperm DNA. The progeny of irradiated mice are then screened for obvious phenotypic abnormalities. Such mutagenesis screens have been very useful in producing novel mutants, but a major problem is that the mutations occur essentially at random. Identification of the structural change(s) in the DNA of a single mutant animal may therefore require a laborious positional cloning approach.

A mutation that is induced by inserting a known foreign DNA sequence (transgene) into the mouse genome has a major advantage over one induced by chemical mutagens or X rays: it leaves a sequence tag at the locus which is mutated. As a result, rapid molecular characterization of the mutated locus is possible. Mouse ES cells provide a way of introducing such mutations into the germ line, and so random insertional mutagenesis in ES cells using transgenes was considered a useful way of producing a large number of mouse mutants which could quickly be characterized at the molecular level. Unlike gene targeting, the transgene should not be related to endogenous sequences, but should integrate essentially at random by nonhomologous recombination. However, because large sections of the mouse genome are noncoding, many random insertion events may not result in gene disruption.

In order to improve the efficiency of recovering mutations that are likely to have a phenotypic effect (by altering a gene or its expression) the gene trap approach was devised (see Evans et al., 1997). The underlying principle is that the transgene which is inserted into ES cells contains a defective reporter gene or marker gene which lacks some component needed for gene expression. The reporter or marker gene is designed to be expressed only after it inserts into a gene (within an intron or exon) or at a promoter. When it integrates at such positions it can acquire the expression element that it lacks.

Different gene trap strategies are possible. In some cases, the reporter lacks a functional promoter and so relies on chance integration next to an appropriate cis-acting sequence element that can activate its transcription. Other approaches have used a marker gene coupled to a suitable promoter but lacking a downstream polyadenylation signal. Here, the marker gene is designed to be expressed after integrating into a host cell gene such that a fusion RNA product is made that utilizes 3′ host sequences in order to acquire a poly (A) tail (see Figure 21.10). Already, this type of approach has been applied on a large scale, with a recent report describing disruption and sequence identification of 2000 genes in mouse embryonic stem cells (Zambrowicz et al., 1998).

Figure 21.10

Gene trapping uses an expression-defective transgene to select for chromosomal integration events that occur within or close to a gene. A specimen host cell gene with 4 exons (E1–E4) is shown at top together with its promoter (P) and polyadenylation (more...)

21.4.1 Animal models of disease occurring spontaneously or as a result of exposure to chemical mutagens or radiation may be difficult to identify

Spontaneous animal disease models

Mutant human phenotypes, especially those associated with obvious disease symptoms, are subject to intense scrutiny: most individuals who suffer from a disorder seek medical advice. If they present with a previously undescribed phenotype their case may well be referred to experts who often will document the phenotype in the medical literature. Given the motivation of both affected individuals and their families, physicians and interested medical researchers, and the large population size for screening (current total global population is ~6 billion individuals), there is a remarkably effective screening process for mutant human phenotypes. In contrast, many animal disease phenotypes will go unrecorded. Only a small percentage of the animal population is in captivity, and recording of spontaneous mutant phenotypes is largely dependent on examination of animal colonies bred for research purposes and, to a lesser extent, livestock and pet populations. Only mutants with obvious external anomalies are likely to be noticed.

Despite the difficulty in identifying spontaneous animal mutants, a number of animal phenotypes have been described as likely models of human diseases (see Table 21.2 for some examples). In some cases, the animal mutant phenotype closely parallels the corresponding clinical phenotype, but in others there is considerable divergence because of species differences in biochemical and developmental pathways (see Erickson, 1989; Erickson, 1996; Wynshaw-Boris, 1996; see also Section 21.4.5). Additionally, phenotypic differences may result because of different classes of mutation at orthologous loci.

Table 21.2

Examples of spontaneous animal mutants.

Random mutagenesis using chemicals and irradiation

Classical methods of producing animal mutants have involved controlled exposure to mutagenic chemicals, notably ethyl nitrosurea (ENU) and ethyl methylsulfonate (EMS) or to high doses of X-rays. Large numbers of Drosophila and mouse mutants have been obtained by this method and, very recently, efficient large-scale mutagenesis screens have been conducted on both mice and also zebrafish (which offers some advantages as an animal model; see Box 21.2). A major problem with chemically-induced and irradiation-induced mutations, however, is that they are generated essentially at random. In order to identify a mutant phenotype of interest, a laborious screen for mutants needs to be conducted by close examination of the phenotypes following mutagenesis. The mutant phenotypes which have been described in these studies, as well as for spontaneous mutants, show a clear bias towards phenotypes with obvious external abnormalities, simply because of the ease of identifying them. Nevertheless, several important models of human disease have been created using such methods.

Box 21.2

The potential of animals for modeling human disease. Primates should provide the best animal models of human disease because they are so closely related to us (see Section 14.6.2). Humans and great apes show extensive developmental, anatomical, biochemical (more...)

21.4.2 Mice have been widely used as animal models of human disease largely because specific mutations can be created at a predetermined locus

Spontaneous and artificially produced disease phenotypes have been described in a wide range of animal species with differing potentials for modeling human disease. In some cases, the species may be too evolutionarily remote from humans to provide useful disease models. For example, numerous Drosophila mutants have been generated and studies of some developmental mutants have enabled the identification of human genes that are important in development, but they cannot serve as useful models of human disease. Other species, such as the zebrafish, which are also evolutionarily remote from us, offer some advantages as model organisms and models have been reported for some human diseases (Box 21.2). Mammals would be expected to provide better disease models but, for a variety of reasons, our closest relatives, the great apes, have not been very useful in providing disease models. Instead, other mammals, notably mice, have been used widely to model human disease (see Box 21.2).

In the case of animal disease models which are artificially induced by exposure to mutagenic chemicals or radiation, or which originate spontaneously, there is little or no artificial control over the resulting phenotype and frequently, the identification of an animal disease model is serendipitous. The great advantage of transgenic/genetargeted mouse models of disease is that specific disease models can be constructed to order. Provided that the relevant gene clones are available, including mutant genes in some cases, mice can be generated with a desired alteration in a chosen target gene. All the major classes of disease, inherited disorders, cancers, infectious diseases and autoimmune disorders can be modeled in this way (Table 21.3; Smithies, 1993; Clarke, 1994; Bedell et al., 1997). In most cases, the transgenic/gene targeting approaches have been used to model single gene disorders but, increasingly, attempts are being made to produce mouse models of complex genetic diseases, such as Alzheimer's disease, atherosclerosis and essential hypertension effects.

Table 21.3

Examples of transgenic or gene-targeted mouse models of human disease.

21.4.3 Single gene disorders resulting from loss of function and gain of function mutations can be conveniently modeled by gene targeting and by integration of mutant genes respectively

21.4.4 Considerable effort is currently being devoted to constructing mouse models of cancers and other complex genetic disorders

21.4.5 Mouse models of human disease may be difficult to construct because of a variety of human/mouse differences

It is not uncommon for spontaneous or artificially generated mouse models of disease to show phenotypes that are considerably different from the homologous human disorders. For example, gene targeting to inactivate several mouse tumor suppressor genes has often produced disappointing mouse models, as in the case of TP53 and RB1 (retinoblastoma) knock-outs. There may be problems in achieving the desired type of mutation. For example, in gene targeting intended null mutations may be offset in some cases by exon skipping or some other form of ‘leaky’ transcription, or the expression of a transgene may be affected by various parameters causing an unexpected phenotype. Setting aside these possibilities, there are several areas where differences between mice and humans could be expected to result in divergent disease phenotypes for mutations in orthologous genes (Erickson, 1989; Erickson, 1996; Wynshaw-Boris, 1996):

• Biochemical pathways. Although biochemical pathways in mammals are generally well conserved, some differences are known between the pathways of humans and mice. The human retina appears to depend heavily on the accurate function of the Rb gene product, but other vertebrate retinas do not. As a result, spontaneous retinoblastoma mouse mutants have not been described, and retinoblastoma is not a feature of Rb1 knock-out mice. Another example may be provided by ganglioside degradation pathways and while deficiency of the hexosaminidase gene HEXA results in the severe Tay-Sachs disorder, inactivation of the mouse homolog Hexa results in abnormal accumulation of ganglioside in neurons but without motor deficits or learning deficits (Wynshaw-Boris, 1996).
• Developmental pathways. The differences in human and mouse developmental pathways are not well understood but are expected to be significant for some organ systems, such as in brain development.
• Absolute time. Because of the huge difference in the average lifespans of mice and humans, certain human disorders in which the disease is of late onset may possibly be difficult to model in mice.
• Genetic background: the importance of modifier genes. Most human populations are outbred. Laboratory strains of mice, however, are very inbred. Often a particular phenotype can vary considerably in different strains of mice because of differences in their alleles at other loci (modifier genes), which can interact with the locus of interest.

A useful example of the importance of genetic background is the Min (multiple intestinal neoplasia) mouse which was generated by ENU mutagenesis and results from mutations in the mouse Apc gene. Mutations in the orthologous human gene, APC, cause adenomatous polyposis coli and related colon cancers and the Min mouse has been regarded as a good model for such disorders. The phenotype of the Min mouse is, however, dramatically modified by the genetic background. For example the number of colonic polyps in mice carrying APC^Min is strikingly dependent on the strain of mouse. Similar phenotypic variability is found in human families where different members of the same family may have strikingly different tumor phenotypes although they possess identical mutations in the APC gene. Some of the variability could be due to environmental factors, but the involvement of modifier genes had been strongly suspected. The Min mouse provides a well-defined genetic system for mapping and identifying modifier genes (Dietrich et al., 1993; MacPhee et al., 1995).

21.5.1 Principles and practice of animal cloning

The term clones indicates genetic identity and so can describe genetically identical molecules (DNA clones), genetically identical cells or genetically identical organisms. Animal clones occur naturally as a result of sexual reproduction. For example, genetically identical twins are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of animal cloning can also occur as a result of artificial manipulation to bring about a type of asexual reproduction. The genetic manipulation in this case uses nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed. The resulting ‘renucleated’ oocyte can give rise to an individual who will carry the nuclear genome of only one donor individual, unlike genetically identical twins. The individual providing the donor nucleus and the individual that develops from the ‘renucleated’ oocyte are usually described as ‘clones’, but it should be noted that they share only the same nuclear DNA; they do not share the same mitochondrial DNA, unlike genetically identical twins.

Nuclear transfer technology was first employed in embryo cloning, in which the donor cell is derived from an early embryo, and has been long established in the case of amphibia. However, it was only comparatively recently when McGrath and Solter reported nuclear transplantation in the mouse embryo and paved the way for modern mammalian cloning (see Fulka et al., 1998). Subsequently, nuclear transplantation was conducted successfully in the eggs of domestic animals, including sheep and cows.

Unlike embryo cloning the prospect of cloning adults had seemed remote. During their development from (ultimately) the fertilized oocyte, adult somatic cells undergo an extensive series of cell division and differentiation steps. Until recently it was thought that these processes were accompanied by irreversible modifications to the genome. Even in frogs transplantation of an adult cell nucleus had never been reported to give rise to an adult animal; instead, the renucleated embryos underwent early development but thereafter failed to develop to term. A variety of early experiments in mice were also unsuccessful before the landmark study of Wilmut et al. 1997 reported successful cloning of an adult sheep. For the first time, an adult nucleus had been reprogrammed to become totipotent once more, just like the genetic material in the fertilized oocyte from which the donor cell had ultimately developed.

In the Wilmut et al. study, the donor cells were derived from a cell line established from adult mammary gland cells and were fused to an enucleated metaphase II-arrested oocyte (Figure 21.11A). The donor cells were deprived of serum before use, forcing them to exit the cell cycle into a quiescent stage, G_o (Stewart, 1997). A certain degree of gene silencing is a characteristic feature of the nuclei of G_o cells. As egg cells are normally fertilized by transcriptionally inactive sperm cells, G_o cells may be more amenable to full genetic reprogramming. Another consideration is the degree of chromosome condensation and of access to chromatin ‘remodeling factors’ such as transcription factors in the oocyte. In any event, the cloning was extremely inefficient: out of a total of 434 oocytes that were submitted to the procedure, only 29 developed to the transferable stage and of these only one developed to term, being born as the now famous Dolly (Figure 21.11B). Subsequent doubts about the exact origin of the donor cell and whether Dolly really was an adult clone (as opposed to a contaminant fetal cell) have been allayed by genetic testing of Dolly and the adult mammary gland donor cells. Importantly, successful animal cloning has also been achieved by other groups with comparatively high success in cloning of adult mice (Wakayama et al., 1998) and cows (Kato et al., 1998).

Figure 21.11

A sheep called Dolly was the outcome of the first successful attempt at animal cloning. (A) Experimental strategy used by Wilmut et al. (1997). During culture in serum-depleted medium the normal cell cycle is interrupted and cells move from the G₁ phase (more...)

21.5.2 The successful cloning of an adult animal has major implications for research, medicine and society

The report by Wilmut et al. (1997) has generated enormous attention, in the scientific and general press, both because of its novelty and the significance for future work. In particular, the possible extrapolation to cloning of humans has generated a great deal of controversy.