Journal
GENESIS
Volume 61, Issue 1-2, Pages -Publisher
WILEY
DOI: 10.1002/dvg.23511
Keywords
mammal; organism; reproductive; tissue
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This study describes the generation of the Ddx4-P2A-iCre allele through CRISPR/Cas9 targeted insertion of an improved Cre sequence. Functional assessment of this allele indicates that Cre activity begins in primordial germ cells from at least E10.5 and achieves higher efficiency for early gonadal deletion compared to the inducible Oct4(CreERT2) line. The Ddx4(iCreJoBo) allele shows hypomorphic Ddx4 expression and infertility in homozygous males, with ectopic activity observed in multiple organs.
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.
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