Non‐parallel recombination limits cre‐loxP‐based reporters as precise indicators of conditional genetic manipulation

Abstract

Cre/LoxP-mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage-tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre-expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre-mediated recombination, which raises concerns regarding utilization of common Cre-reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre-expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non-parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent-target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations.

PDGFRβ-P2A-CreERT2 mice: a genetic tool to target pericytes in angiogenesis

“…However, we observed that efficiency of recombination in pericytes using this reporter was less than that observed using the td-Tomato reporter, in this case 41.94 ± 18.67 % (n=5) of NG2 + cells were GFP + . Discrepancies between recombination efficiency in different mouse reporter lines (including Rosa-tdTomato and Rosa-mT/mG ) have already been described by Liu et al [19], and the differences we observed in efficiency are similar to what has been previously noted. Liu et al proposed that their results imply that reporter sensitivity inversely correlates with the distance between the LoxP sites, a theory that is consistent with previous observations by Coppoolse et al [20].…”

Six3 in a small population of progenitors at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice

“…S8). It was reported that non-parallel recombination exists between R26R reporter and targeted gene deletion, and the differences in the chromosomal location, the distance spanning LoxP sites, the sequences around the LoxP sites and the level of Cre activity in the cells probably contribute to the non-parallel recombination (Liu et al, 2013). In our hand, Six3-deletion and β-gal reporter expression are mostly exclusive and we did not see complete Six3-deletion in E8.5 Six3 mutant embryos in which drastic phenotypes were obvious, no matter Six3 -deletion was driven by Six3-Cre (Fig.…”

Lineage fate of ductular reactions in liver injury and carcinogenesis

“…Different CreER strains may exhibit highly variable sensitivity to tamoxifen-induced reporter expression, depending on the strength of the Cre-driving promoter and the respective reporter strain used (34). Further, the possibility of tamoxifen-independent reporter expression as well as de novo recombination events caused potentially by tamoxifen that was not eliminated during injury must be considered.…”