Mouse interspecific backcross (IB) linkage maps provide almost limitless variation for gene mapping. Using an IB composed of 205 [(C57BL/6J X Mus spretus) X C57BL/6J] progeny, we have developed a high-resolution molecular genetic linkage map of the mouse genome that includes more than 3000 loci and covers most of the known mouse genetic map. This map has facilitated the genetic studies of many scientists throughout the world in addition to scientist in our own Program at NCI-Frederick. In collaboration with Dr. Donald Court at NCI-Frederick, we have developed a highly efficient phage-based Escherichia coli homologous recombination system that makes it possible to modify genomic DNA in bacterial artificial chromosomes (BACs) and to subclone genomic DNA from BACs into multicopy plasmids without the need for restriction enzymes or DNA ligases. Virtually any kind of mutation can now be engineered into BACs without the need for a confounding linked drug selection marker including point mutations, insertions, and deletions. This new form of chromosome engineering, termed """"""""recombineering"""""""", is very efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiments that were difficult or impossible to perform by earlier methods and should enhance functional genomic studies in the postgenomic era, by allowing the creation of better mouse models and a more refined genetic analysis of the mouse genome. FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila melanogaster and for discerning the function of recessive genes in a heterozygous individual. In studies that are in press in Nature Genetics, we have found that mitotic recombination can also be reproducibly induced in mouse ES cells with Cre/loxP at a frequency of 4.2 X 10-5 (Snrpn) to 7.0 X 10-3 (D7Mit178) for single allelic loxP sites following transient Cre expression. We have also found that the frequency of mitotic recombination can be increased with multiple loxP sites (5.0 X 10-2, D7Mit178) and by constitutive Cre expression. Interestingly, much of the recombination occurs in G2 and is followed by X segregation where the recombinant chromatids segregate away from each other during mitosis. X segregation is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm those of Beumer and colleagues predicting that X segregation will not be limited to organisms like Drosophila with strong mitotic pairing because the forces responsible for X segregation are an elemental feature of mitosis in all eukaryotes. They also indicate that genetic mosaic analysis is feasible in mice, at least for certain chromosomal regions.
| Dave, Utpal P; Jenkins, Nancy A; Copeland, Neal G (2004) Gene therapy insertional mutagenesis insights. Science 303:333 |
