Question

Is light coat color in beach mice and rock pocket mice homologous or analogous? Explain your reasoning, including a discussion of the genetic control of the trait.

Answer 1

The process of gene targeting provides a means to alter a specified gene in order to better discern its biological role. Through homologous recombination, an engineered mutation can be directed to a designated genetic locus. In this manner, a potentially important genomic clone can directly be utilized to create a mutation into a selected gene. Even amongst the 2.5 Gb of the mouse genome, the cellular DNA repair mechanisms are able to align a targeting vector with its corresponding region of homology and cause recombination into the chromosome. While the goal of transgenic technology is to overexpress a gene to study its biological role in vivo, homologous recombination is typically employed to create a ‘loss of function’ mutation. The most common application of gene targeting is to produce knockout mice, where a drug resistance marker replaces an essential coding region in a genetic locus. In the majority of cases, the importance of a gene cannot be determined by simply recognizing amino acid motifs in the protein (Iredale, 1999). Additionally, the role of a gene often cannot be completely revealed by examining closely related family members. So, gene inactivation is the best way to delineate the biological role of a protein and gene targeting is a direct means to disrupt a gene’s open reading frame and block its expression in a mouse. Not surprisingly, during the twenty years that gene targeting techniques have been available, thousands of genes have been knocked out. To date, about 11,000 genes have been knocked out in mice, which accounts for roughly half of the mouse genome (Vogel, 2007; Sikorski and Peters, 1997). Through a combination of gene targeting and gene trapping, a global effort is underway to make a knockout mouse for all of the 25,000 mouse genes (Grimm, 2006).

The knockout mouse has been a valuable tool for geneticists to discern the role of a gene in embryonic development and in normal physiological homeostasis. Mice act as a good analogue for most human biological processes since both species share about 99% of the same genes (Capecchi, 1994). Additionally, mice are useful experimental animals because they are small, have relatively short life spans, and are prolific. So, for geneticists, the targeted deletion of a gene in a mouse provides an important means to determine the biological role of a genetic allele. While useful to study in vivo gene function, some knockout mice have also additionally served as valuable animal models for human genetic diseases. When a human mutation is found to disable a protein, the corresponding knockout mouse can be an important resource to study the underlying pathophysiology and to develop therapies to treat a genetic disease (Majzoub and Muglia, 1996). Additionally, pharmaceutical companies obtain clues about inhibiting a protein by first looking at the phenotype of a knockout mouse (Zambrowicz and Sands, 2003). Thus, knockout mice can provide insight into a gene’s physiological role in humans.

Rather than just inactivate a gene, however, some genetic diseases result in the expression of a mutated protein. Point mutations, micro-deletions, or insertions are often responsible for many human genetic diseases. These subtle mutations can also be mimicked in a mouse model using gene targeting. Instead of disrupting a gene, as in most knockout mice, homologous recombination is employed to swap the normal copy of an exon with a mutated version. As long as a similar mutation can be reproduced in the mouse protein, then the corresponding amino acid substitution, deletion, or insertion can be targeted into a gene of interest to replicate the human disease. The effects of the altered protein can then be studied in the animal model.

With some knockout mouse models, the severity of the phenotype can preclude analysis of a gene’s role in the organogenesis of a particular tissue. For example, about 15% of all knockout mice have mutations that result in developmental lethality (www.genome.gov/12514551). To circumvent this problem, Cre/loxP technology has been employed to create conditional knockout mice. Derived from the P1 bacteriophage, the Cre recombinase will excise any region of DNA placed between two loxP sites (locus of X-ing over) (Sauer and Henderson, 1998; Sternberg and Hamilton, 1981). The loxP site is a 34 bp nucleotide sequence that can be genetically targeted around an essential exon in a gene (Gu et al., 1994). The resulting mice contain the floxed (flanked by loxP sites) allele in all tissues but are phenotypically wildtype. These floxed mice are then bred to Cre expressing transgenic animals, wherein the promoter used to drive Cre expression will determine the site of the gene deletion. For spatial inactivation of a gene in a mouse, a cell type specific promoter is used to limit Cre expression to a particular tissue. Although less commonly used than Cre/loxP technology, the Flp recombinase also provides a similar means to rearrange a genetic locus. Flp (flippase) was isolated from Saccharomyces cervisiae and, like Cre, the recombinase will also excise DNA flanked between 34 bp sequences known, in this case, as FRT sites (Dymecki, 1996). So, through the use of either the Cre/loxP or the Flp/FRT systems, gene expression can be disrupted in a spatial and temporal manner and the lethality of a knockout mouse phenotype can be overcome. With this versatility, mice utilizing Cre/loxP or the Flp/FRT systems are often shared amongst research laboratories studying differing physiological systems.

In addition to spatial excision of a floxed allele, temporal control of Cre-mediated recombination is also possible in a conditional knockout mouse. The timing of recombination can be regulated by use of a tamoxifen-inducible Cre (Feil et al., 1997; Hayashi and McMahon, 2002). In this strategy, Cre is ligated to a mutated ligand binding domain of the estrogen receptor that restricts transcription until tamoxifen is present. Cre is, therefore, not expressed until tamoxifen is applied either topically or through injection. Another inducible Cre system takes advantage of the reverse tetracycline-controlled transactivator (rtTA) (Utomo et al., 1999). Doxycycline is administered to activate the rtTA that, in turn, will induce transcription of Cre. Lastly, Cre can also be delivered through injection of a viral vector (Anton and Graham, 1995). When and where the Cre is expressed is controlled by the timing and site of injection for the virus. The amount of DNA rearrangement can be adjusted by varying the titer of the virus. The numbers of conditional knockout mice have dramatically increased since Cre/loxP and Flp/FRT technologies were introduced into gene targeting. This proliferation of conditional animal models attests to the value of the recombinases as a molecular switch.

Another interesting application of gene targeting is knock-in technology, in which any gene of interest can be placed under the cis-acting regulatory elements of another gene (Cohen-Tannoudji and Babinet, 1998). Originally, knock-in mice were derived as a means to visualize a gene’s expression due to targeted recombination of the lacZ reporter gene into a genetic locus (LeMouellic et al., 1990). The initial coding sequence of a gene would be replaced with the lacZ marker that is inserted under the direction of the gene’s promoter. The strategy of using homologous recombination to knock-in a reporter gene, like lacZ, allows for not only the creation of homozygous null mice for a gene, but also provides a technique to study the targeted gene’s expression in the heterozygous mice that are often phenotypically normal. The knock-in idea was later elaborated to include the replacement of a gene for the sequence of a similar isoform of the protein (Hanks et al., 1995). Therefore, similar protein isoforms could be tested for redundancy. Through knock-in mice, genetically similar proteins can be examined to determine if two isoforms are truly biologically distinct or if the proteins are basically functionally equivalent with differing patterns of expression being the only key divergence.