Generation and Analysis of Partially Haploid Cells with Cre-mediated Chromosome Deletion in the Lymphoid System

Abstract

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. Over 4000 protein-coding genes have been mutated and studied in the mouse by gene targeting, gene trapping, or other mutagenic methods in the past 2 decades (1, 2). This number is expected to increase dramatically following the recent calls for genome-scale coverage of germ line mutations by several international consortia (3). Studies of the existing germ line mutations have implied that approximately one-third of null mutations cause embryonic or neonatal lethality (4). Consequently, the effect of germ line mutations on postnatal life cannot be easily assessed for the genes playing important roles in both embryonic and postnatal life. Further analysis of tissue-specific gene function often requires a completely independent effort to establish a new allele for Cre/ lox-mediated conditional gene knock-out. Although the Cre/lox system can effectively address the embryonic problem and permit the study of tissue-specific gene function, the experimental system is not practical for large-scale genetic analysis. Thus far, there is no simple method available for systematic evaluation of lymphoid phenotypes of lethal mutations accumulated from a large-scale mutagenesis approach.Mitotic recombination provides one possible way to assess somatic phenotypes of pre-existing mutations. A recombination between homologous chromosomes during mitosis can lead to clonal segregation of heterologous alleles. The mosaics resulting from mitotic recombination allow for functional analysis of homozygous clones in an otherwise heterozygous background (5, 6). The feasibility of mosaic analysis in mice was demonstrated in the lo...

Whole chromosome loss in tetraploid cells confers tumorigenic potential in a mouse allograft model

“…Upon Cre expression, recombination between the duplicated sister chromatids yields either the same parental configuration or an inverted recombination to yield a pair of acentric and dicentric fragments ( Figure 1B). The acentric fragments are not maintained during repeated cell divisions and the dicentric fragments undergo breakage-fusion-bridge cycles in subsequent mitoses and are eventually lost (Zhu et al, 2010).…”

“…For brevity, we refer to cells with engineered chromosome loss events as ICLs (cells with Induced-Chromosome Losses) and their unrearranged parental counterparts as controls. Previous adaptation of this strategy, in the context of studying loss of heterozygosity (LOH), has shown that recombinationmediated marker loss also correlated strongly with chromosome loss in the lymphoid system in mice (Zhu et al, 2010), suggesting that marker loss is a faithful measure of the predicted chromosome loss event both in vitro and in vivo.…”

Tracking Proliferative History in Lymphocyte Development with Cre-Mediated Sister Chromatid Recombination

“…Cre recombinase has been shown to be capable of driving recombination between sister chromatids via duplicated loxP sites [11], [12]. This recombination system provides an opportunity for Cre mediated activation of a genetic marker during cell cycle.…”

“…To test the efficiency of sister chromatid recombination, we isolated cells stably expressing GFP and transduced these cells again with a Cre-expressing retroviral vector. Efficiency of transduction was monitored with a hCD2 marker co-expressed with Cre on the retroviral vector [12]. Without cell cycle synchronization, the doubling time for NIH3T3 cells has been reported to be approximately 17 hours [15].…”

“…This system is based on the fact that Cre/lox mediated recombination can occur between sister chromatids during cell cycle [11], [12]. Cre-mediated sister chromatid exchange occurs at a much higher frequency than Cre-mediated mitotic recombination between homologous chromosomes [12], [13]. In our design, a non-equal exchange between the marked sister chromatids produces a fraction of progeny that acquire the fluorescent marker.…”