Overview of HLBS Center Mutagenesis and Genetic Screening Strategies
The goal of mutagenesis for functional genetic studies is to increase the frequency and variety of mutations that will be recovered in a phenotype-driven screen. In the mouse, the chemical N-ethyl-N-nitrosourea (ENU) induces point mutations at a high rate permitting efficient systematic phenotype-based surveys of gene function. As a point mutagen ENU can induce a series of alleles leading to amorphic/null (loss-of-function), hypomorphic (partial loss-of-function), antimorphic (opposing or dominant negative function) and hypermorphic (gain-of-function) phenotypes. ENU-induced mutations also serve as valuable reagents for studies relating protein structure and function.
Screening for models of human disease using chemical mutagenesis of mice
In order to generate new mouse models of human disease the HLBS Center is using ENU to mutagenize C57BL/6J mice. Differences in their genetic background may also serve to sensitize specific inbred strains, making certain strains more likely to uncover a phenotype following a mutation induced by ENU. Therefore, additional strains may be mutagenized as phenotype information is gathered in the accompanying HLBS Center strain characterization project. Mouse models will be recovered using a genetic strategy to survey the entire genome for recessive mutations. A genome-wide recessive screen requires the production and phenotyping of families of third-generation (G3) offspring from a G1 founder (Figure 1). The G1 founder represents the equivalent of one mutagenized gamete (genome) captured in the mating of a wild-type female to an ENU-treated G0 male. Thus, in a genome-wide recessive screen the two critical classes are the G1 mice (representing the number of mutagenized genomes screened), and the G3 mice (representing the number of animals channeled through phenotyping). In the backcross breeding scheme shown in Figure 1, all of the G3 progeny will be recessive for a subset of the mutations induced in the G1 founder with any specific mutation represented in 1/8 of the G3 offspring (also see Figure 2). The planned production of 20 G3 progeny for each G1 founder provides a 92% chance that a new mutation will be seen at least once and a 71% chance that it will be seen two or more times. In addition to revealing a phenotypic deviant this strategy provides valuable insight on potential heritability. Dominant mutations will also be detected when phenotyping the G3 population. The HLBS Center will produce and screen ~5,000 G3 mice per year. A total of 250 mutagenized genomes will be surveyed each year by phenotyping 20 G3 mice from each G1 founder.
Screening for models of human disease using chemical mutagenesis of mouse ES cells
Mouse embryonic stem (ES) cells can contribute to all tissues during development, including the germline, permitting genetic modifications to be introduced in culture and analyzed in the mouse. Using mouse ES cells to combine advantages inherent to cell culture systems with chemical mutagenesis greatly enhances genetic approaches to address gene function. The ability to modulate and readily assay mutation rate, improved quality control, higher mutation frequency, a wider spectrum of mutagens and shortened breeding strategies in mice are attractive features of ES cell mutagenesis.
Mouse ES cells can be effectively mutagenized and used to generate mice following treatment with the point mutagens ENU and ethylmethanesulfonate (EMS, another alkylating agent commonly used in flies and worms). ES cell mutagenesis permits a genome-wide recessive screen to be performed in two rather than three generations (Figure 2). In the scheme shown, chimeric male mice derived from injecting blastocysts with chemically mutagenized ES cells are used to initiate the screen. The chimeric males are mated to wild-type females, and the G1 females are backcrossed to the original chimera. Alternatively, the G1 mice can be intercrossed to generate offspring that potentially carry recessive mutations (scheme not shown). In the breeding protocol shown 1/4 of the G2 mice will be homozygous for a mutation at a given locus, increasing the opportunity to identify a mutant as the G2 mice are channeled through the series of tests to detect a phenotype.
ES cell mutagenesis can produce twice as many mutants in two generations compared with a traditional whole-animal ENU three generation recessive breeding scheme.
Program for Genomic Applications (PGA)
Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611)
