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There are two problems inherent in all methods of classical mutagenesis. The first problem is that the process is entirely random. Thus, one must start out by designing a screening assay to allow the detection of mutations at the locus of interest, and then one must hope for the appearance of mutant animals at a frequency which is at, or above, the usual per-locus rate. If a mutant allele fails to produce a phenotype that can be picked up by the screen, it will go undetected. Finally, even when mutant alleles are detected, the underlying lesion can usually not be ascertained without cloning and further molecular characterization.

The second problem with classical mutagenesis is that induced mutations are not tagged in any way to provide a molecular entry into a locus that has not yet been cloned. Thus, if a novel locus is uncovered by an induced mutation that causes an interesting phenotype, it can only be approached through candidate gene and positional cloning approaches in the same way as any other phenotypically defined locus. Furthermore, in the case of ENU-induced mutations, the mutant and wild-type alleles are likely to be molecularly indistinguishable with the exception of a single nucleotide that may or may not affect a restriction site.

One can imagine two types of mutagenic approaches that would be most ideal for the two different types of situations in which mutations can provide tools for molecular analysis of development and other aspects of mammalian biology. On the one hand, a random mutagenesis approach is fine for the elucidation of novel loci so long as the mutant allele is tagged to allow direct molecular access. On the other hand, to further analyze a locus which is already cloned and characterized, one would like to generate animals that misexpress the locus in some defined manner. The technologies of transgene insertion and gene targeting have provided geneticists with the tools needed to accomplish both of these goals.

6.3.1 Overview

In 1981, five independent laboratories reported the insertion of foreign DNA into the mouse germ line through the microinjection of one-cell eggs (Costantini and Lacy, 1981; Gordon and Ruddle, 1981; Harbers et al., 1981; Wagner et al., 1981a; Wagner et al., 1981b). Although the incorporation of exogenous DNA into the germ line through viral infection of embryos had been reported earlier (Jaenisch, 1976), the 1981 reports implied for the first time that DNA from any source could be used to transform the mouse genome. The complexion of mouse genetics was changed forever with the development of this powerful tool. A strictly observational science was suddenly thrust into the realm of genetic engineering with all of its vast implications. The insertion of genetic material into the mouse germ line has now become sufficiently routine that the methodology is detailed in various "cookbooks" (Wassarman and DePamphilis, 1993; Hogan et al., 1994) and designer animals are even provided as a commercial service by a number of companies.

The term transgenic has been coined to describe animals that have foreign sequences inserted stably into their genome through human intermediaries. Transgenic animals can be created by microinjection or viral infection of embryos, or through the manipulation in culture of embryonic-like "ES cells" that are subsequently incorporated back into the embryo proper for shepherding into the germ line. The latter technology will be discussed in a following section. Here I will focus on transgenic animals created by direct injection of DNA into embryos.

The initial animal that develops from each micro-manipulated egg is called a founder. Even when multiple embryos have all been injected or infected with the same foreign DNA, the integration site — or transgene locus — in each founder will be different. However, all transgenic animals that descend from a single founder will share the same transgene locus. Protocols for the creation of transgenic mice, and extensive reviews of the technology and its uses have been described elsewhere (Palmiter and Brinster, 1986; Wassarman and DePamphilis, 1993; Hogan et al., 1994). Rules for naming transgene loci and transgenic animals are presented in Section 3.4.

With current protocols for the creation of transgenic mice by embryo microinjection, the site of integration is not predetermined, and, for all practical purposes, should be considered random. Microinjection allows one to add, but not subtract genetic material in a directed manner; if a particular experiment leads to the insertion of a novel version of a mouse gene into the genome, this novel allele will be present in addition to the normal diploid pair. Consequently, only dominant, or co-dominant, forms of phenotypic expression will be detectable from the transgene.

The embryo microinjection technology can be used to explore many different aspects of mouse biology and gene regulation. One class of experiments encompasses those aimed at determining the effects of expressing a natural gene product in an unnatural manner. By combining the gene of interest with regulatory regions chosen from other genes, one can cause transgenic mice to express the product at a higher than normal level, or in alternative tissues or developmental stages. The mutant phenotypes that result from such aberrant forms of expression can be used to elucidate the normal function of the wild-type gene. Experiments of this type can be used, for example, to demonstrate the capacity of some genes to induce specific developmental changes and the oncogenic nature of others when they are expressed aberrantly. Many other types of questions can be answered with this approach.

In another class of experiments, one can dissect out the function of a regulatory region by forming constructs between it and a reporter gene whose expression can be easily assayed in the appropriate tissue(s). With a series of transgenic lines that have partially deleted or mutated forms of a regulatory region, one can pinpoint which DNA sequences are involved in the turning on and turning off of genes in different tissues or developmental stages.

A third of class of experiments is aimed at correcting a genetic defect in a mutant mouse through the genomic insertion of a wild-type transgene. This use of the transgenic technology provides the most powerful means available to prove that a cloned candidate gene is indeed identical to the locus responsible for a particular mutant phenotype. Furthermore, the correction of genetic defects in model mammals is a necessary prelude to any attempt to perform similar studies in humans.

An important consideration in all transgenic experiments follows from the observation that the actual chromosomal location at which a transgene inserts can play a determining role in its expression. This will be readily apparent in cases where different founder lines with the same transgene show different patterns of transgene expression. The reason for such strain-specific differences is that some chromosomal regions are normally maintained in chromatin configurations that can act to suppress gene activity. Different transgene constructs will show different levels of sensitivity to suppression of activity when they land in such regions.

Another potential problem can result from the insertion of the transgene into a normally-functioning endogenous locus with unanticipated consequences. In approximately 5-10% of all cases studied to date, homozygosity for a particular transgene locus has been found to cause lethality or some other phenotypic anomaly. (Palmiter and Brinster, 1986). These recessive phenotypes are most likely due to the disruption of some normal vital gene. In less frequent cases, a transgene may land at a site that is flanked by an endogenous enhancer which can stimulate gene activity at inappropriate stages or tissues. This can lead to the expression of dominant phenotypes that are not strictly a result of the transgene itself. 39 For all of these reasons, it is critical to analyze data from three or more founder lines with the same transgene construct before reaching conclusions concerning the effect, or lack thereof, on the mouse phenotype.

In the vast majority of cases analyzed to date, the disruption of endogenous sequences caused by transgene integration has had no apparent effect on phenotype. However, the absence of a detectable phenotype does not necessarily mean that the transgene has integrated into a non-functional region of the genome. As discussed in Chapter 5, only a small subset of all mammalian genes are actually vital, and subtle effects on phenotype are likely to go unnoticed if one performs only a cursory examination of transgenic animals. Thus, the actual frequency of insertional mutagenesis resulting from embryo microinjection is likely to be significantly higher than the numbers imply.

6.3.2 Tracking the transgene and detecting homozygotes

Unless a particular transgenic insertion causes an easily detectable, dominant phenotype, the presence of the transgene in an animal is most readily determined through DNA analysis. For testing large numbers of mice, the best source of DNA is from tail clippings or ear punch-outs (Gendron-Maguire and Gridley, 1993). The presence or absence of the transgene can be demonstrated most efficiently with a method of PCR analysis that is based on transgene-specific target sequences.

The founder animal for a transgenic line will be heterozygous for the transgene insertion locus. The second homolog will be associated with a non-disrupted "wild-type" (+) allele at this locus, whereas the disrupted chromosome will carry a transgene (Tg) allele. As long as the transgene is transmitted to offspring from a heterozygous (Tg/+) parent, it will be necessary to test each individual animal of each new generation for the presence of the transgene. For this reason alone, it would be useful to generate animals homozygous for the transgene allele since all offspring from matings between homozygous Tg/Tg animals would also be homozygous and there would be no need for DNA testing.

In rare cases, homozygous Tg/Tg animals will be phenotypically distinct from their Tg/+ cohorts. This observation is usually a good indication that the transgene has disrupted the function of an endogenous locus through the process of integration. If the homozygous recessive phenotype is lethal, it will obviously be impossible to generate a homozygous line of animals. Otherwise, the phenotype may eliminate the need for DNA analysis. In the vast majority of cases, however, homozygous Tg/Tg animals will be indistinguishable in phenotype from heterozygous Tg/+ animals, and without a recessive phenotype, the identification of homozygous animals will not be straightforward.

One approach to confirming the genotype of a presumptive Tg/Tg animal is based on statistical genetics. In this case, confirmation is accomplished by setting up a mating between the presumptive homozygote and a non-transgenic +/+ partner. If the animal in question is only a Tg/+ heterozygote, one would expect equal numbers of Tg/+ and +/+ offspring. Through the method of chi-square analysis described in Section 9.1.3, one can calculate that if at least 13 offspring are born and all carry the transgene, the probability of a heterozygous genotype is less than one in a thousand. 40 If even a single animal is obtained without the transgene, the parent’s genotype will almost certainly be Tg/+. Statistical testing of this kind must be performed independently for each presumptive Tg/Tg animal. Once homozygous Tg/Tg males and females have been confirmed, they can be re-mated to each other as the founders for a homozygous transgenic strain.

A second approach to demonstrating transgene homozygosity requires the cloning of an endogenous sequence from the mouse genome that flanks the transgene insertion site. This task is often not straightforward because transgenic material can be present in multiple copies that are intermingled with locally rearranged endogenous sequences. Nevertheless, with the cloning of any nearby endogenous sequence, one obtains a mapping tool that, in theory, can be used to distinguish both the disrupted and nondisrupted alleles at the transgene locus through the use of one of the various techniques described in Chapter 8 (8.2, 8.3) for detecting "codominant" DNA polymorphisms. With such a tool, and an associated assay, homozygosity for the transgene allele would be demonstrated by the absence of the wild-type nondisrupted allele. Unfortunately, this approach would require the generation of a separate endogenous clone for each and every transgenic line to be studied. Protocols for locating the transgene insertion site within the mouse linkage map are discussed in Section 7.3.2.

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