Incomplete cre‐mediated excision leads to phenotypic differences between Stra8‐iCre; Mov10l1^lox/lox and Stra8‐iCre; Mov10l1^lox/Δ mice

Abstract

In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deletor line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes due to mosaicism (i.e. co-existence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-iCre, a transgenic allele expressing codon-improved Cre recombinase (iCre) under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (100% inactivation) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; genelox / Δ genotype should be used to evaluate phenotypes instead of Cre; genelox/lox due to the fact that the latter usually bears incomplete deletion of the flox allele(s).

RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development

“…Consistent with incomplete Cre-mediated recombination when multiple floxed alleles are present (48), the level of residual Ptbp2 was elevated (ϳ10% compared to WT) in cKO IRG ϩ testes compared to cKO testes lacking the IRG transgene ( Fig. 5B and C).…”

“…A possible explanation for this difference is that the IRG locus contains an estimated 7 to 10 copies of the IRG cassette (46). Thus, even under conditions of reduced overall recombination activity when multiple floxed alleles are present (48), the high copy number of the IRG cassette permits efficient labeling of germ cells with GFP.…”

Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice

“…To further explore potential roles of MIWI2 in the meiotic and haploid phases of spermatogenesis, we crossed Miwi2 loxp mice with three germ cell-specific Cre lines to inactivate Miwi2 in the male germ line at different developmental time points. The Ddx4-Cre line has been used to delete a floxed allele in PGCs/prospermatogonia with an onset of CRE expression at E15.5, 39 whereas Stra8-Cre mice display CRE expression in prospermatogonia at P3 although the full penetrance of CRE activity is not reached until pachytene spermatocytes at P12 10,40,41 ( Figure 1d). The Prm-Cre line has Cre expression starting in round spermatids, but the protein expression may be delayed due to posttranscriptional regulation 42 ( Figure 1d).…”

“…Hematoxylin-Eosin (HE) staining procedure for paraffin-embedded sections were performed as described before. 41 For immunohistochemistry, GCNA antibody (a kind gift from Dr. Enders Mark) was used at a dilution of 1 : 10, and all steps were performed according to the protocol provided in the commercial ABC kit (Vector Laboratory, Burlingame, CA, USA). For quantitative analyses of GCNApositive germ cells, only round or nearly round cross sections of seminiferous tubules were selected for counting and a total of at least 20 cross sections were counted for each time point.…”

Cell‐specific ablation in the testis: what have we learned?

“…To date this approach combines the fastest clearance of all germ cells with the lowest off‐target toxicity although this approach does rely on the presence of multiple transgenes, which is impractical in most contexts. Furthermore, because of the variable nature of the Cre used (Bao et al ., 2013), a small number (<1%) of spermatogonia fail to express the receptor and thus remain in the testis. However, the fact that these residual cells are able to repopulate the testis to produce complete spermatogenesis, demonstrates there is no long‐term impact of germ cell ablation on testis function using this approach (Rebourcet et al ., 2014a,b).…”