Generation and analysis of partially haploid cells with Cre-mediated chromosome deletion in the lymphoid system.
The fast accumulation of mutant mouse strains in recent years has provided an invaluable resource for phenotype-based genetic screens. However, study of lymphoid phenotypes can be obscured or impractical if homozygous mutations cause early embryonic defects. To aid phenotype screening of germ line mutations in the lymphoid system, we developed a method to induce loss of heterozygosity (LOH) in developing lymphocytes through chromosome deletion. Chromosome deletion was triggered by Cre/loxP-mediated inverse sister chromatid recombination in the G(2)/M phase of the cell cycle, leading to the generation of daughter cells missing part of or the entire recombinant chromosome. We show that the resulting cells were viable and capable of additional rounds of cell division, thus providing raw materials for subsequent phenotypic assessment. We used the recombination system to induce LOH at the E2A locus in developing B cells. A significant loss of pro-B and pre-B cells was observed when the wild-type allele was removed by chromosome deletion from the E2A heterozygous mice, a result consistent with the required role for E2A in B cell development. We also demonstrated the effectiveness of Cre-mediated chromosome deletion in the LOH assay for HEB function in T cell development. Thus, the Cre-mediated chromosome deletion provides a new and effective method for genome-wide assessment of germ line mutations in the lymphoid system.
Zhu
Yi
Y
Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kim
Young-Mi
YM
Li
Shibo
S
Zhuang
Yuan
Y
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002872:Chromosome Deletion; D018095:Germ-Line Mutation; D006238:Haploidy; D019426:Integrases / Q000378:metabolism; D019656:Loss of Heterozygosity; D008208:Lymphatic System / Q000166:cytology*; D051379:Mice
C045073:Cre recombinase; D019426:Integrases