9th Annual Workshop, October 28-31, 1999
Co-sponsored by the U.S. Department of Energy
Development of an embryonic stem cell library of defined mutations
Geoffrey G. Hicks
Manitoba Institute of Cell Biology Centre for Mammalian Functional Genomics, Winnipeg, Canada
Most mammalian genes will soon be characterized as cDNA sequences with little information as to their function. To utilize this sequence information for large-scale functional studies, we developed a process of tagged-sequence mutagenesis to disrupt genes expressed in mouse embryo-derived stem (ES) cells and to characterize each mutation by direct DNA sequencing. Comparison of these sequence tags (PSTs) with the existing databases identifies disruptions of known genes or genes which may be related by homology or functional domains. The process will generate large numbers of insertion mutations that will be available for transmission into the mouse germline where mammalian gene function can be directly analyzed in vivo.
We have recently reported the results from over 400 such mutations (Nature Genetics 16:338). Analysis of this group has substantiated two important assumptions about this sequenced-based approach: 1) The number of target genes is large and approaches the total number of expressed genes; and 2) The PSTs provide enough sequence information to identify disruptions in known genes when compared to the rapidly expanding nucleotide databases. In addition, the analysis has revealed new insights into the mechanisms of gene entrapment and new vector designs.
The ability to induce, characterize and maintain mutations in ES cells circumvents many limitations associated with conventional mammalian genetics, and will greatly increase the number of mutant alleles (typically loss of function mutations) by which gene functions can be studied in mice and in cell lines derived from such mice. The PSTs allow sequence-based screening of the library of mutations, thereby bridging the gap between gene sequence and mammalian gene function. The process will facilitate a functional analysis of a mammalian genome and will provide animal models for human genetic diseases.