Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Generation of radiation-induced deletion complexes in the mouse genome using embryonic stem cells.

As the genetic and physical mapping stage of the Human Genome Project nears completion, the focus is shifting toward the development of technologies for high-throughput analysis of gene function. Whereas DNA sequencing will enable the assignment of presumed function to a large number of genes in mice and humans, it is clear that the great majority of genes will have to be evaluated in vivo to accurately assess their role in a complex organism. While gene targeting in mouse embryonic stem (ES) cells is the current method of choice for the characterization of gene function in mice, it remains relatively labor intensive and lacks the throughput required for analysis of genome function on a large scale. Alternative methods of efficient mutagenesis will clearly be required for this task. Chromosomal deletions are powerful tools in the genetic analysis of complex genomes, enabling the systematic identification and localization of functional units along defined chromosomal regions. Not only are deletions useful for the identification of genetic functions, but they serve as mapping reagents for existing mutations or traits. While their use has been an essential tool in Drosophila genetics, classical mutagenesis in mice has been logistically impractical for generating deletions. We have previously described an efficient method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. In this article, we detail the methodological aspects of this technology and describe the applications of chromosomal deletions for characterizing gene function in ways that make optimal use of the information generated by the first stage of the Genome Project.

Mutations of the mouse Twist and sy (fibrillin 2) genes induced by chemical mutagenesis of ES cells.

A prior phenotype-based screen of mice derived from ethylmethanesulfonate-mutagenized embryonic stem cells yielded two mouse limb defect mutants. Animals heterozygous for the polydactyly ems (Pde) mutation display preaxial polydactyly of the hindlimbs, and homozygous syndactyly ems (sne) animals are characterized by a fusion of the middle digits of their hindlimbs and sometimes forelimbs. We now report that Pde is a new allele of the basic helix-loop-helix protein gene Twist. Sequencing the full-length cDNA and several hundred basepairs of genomic DNA upstream of the coding region failed to reveal a mutation, suggesting that the lesion may be in a regulatory element of the gene. sne is a new fused phalanges (fp) allele of the shaker-with-syndactylism deletion complex (sy), and we show that the genomic lesion is a small deletion removing an entire exon, coincident with the insertion of the 3' end of a LINE element belonging to the TF subfamily.