Generation and characterization of a Ddx4-iCre transgenic line for deletion in the germline beginning at genital...

Germline-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 Ddx4^iCreJoBo , 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 Oct4^CreERT2 line. We found the Ddx4^iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26R^LacZ and R26R^tdTomato ) and a floxed gene of interest (Cripto^flox ) 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 Ddx4^iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4^iCreJoBo line as useful for germline studies in which early gonadal deletion is required.