Generation and characterization of a Ddx4‐iCre transgenic line for deletion in the germline beginning at genital ridge colonization

Abstract

SummaryGermline‐specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole‐body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved Cre (iCre) sequence in‐frame at the 3′ end of the Ddx4 locus to generate the Ddx4‐P2A‐iCre allele. Our functional assessment of this new allele, designated Ddx4iCreJoBo, reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5–12.5) germline deletion when compared to the inducible Oct4CreERT2 line. We found the Ddx4iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26RLacZ and R26RtdTomato) and a floxed gene of interest (Criptoflox) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for Ddx4iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4iCreJoBo line as useful for germline studies in which early gonadal deletion is required.