Specific induction of ablation of chicken primordial germ cells using iCaspase9

【Objective】The purpose of the study was to prepare chicken germ cell lines that express iCaspase9 specifically in primordial germ cells(PGCs),to improve the reproductive transmission efficiency of exogenous PGCs,and to lay the foundation for the production of male or female receptor chicken embryos that induce infertility.【Method】The iCaspase9 fragment was integrated into the chicken genome using the piggyBac transposon,and a stable CAG-iCaspase9mCherry DF-1 cell line transfected with the iCaspase9 fragment was constructed. Its cell ablation effect under the induction of substrate B/B homodimerizer by the iCaspase9 system was identified by fluorescence microscope. Using CRISPR/Cas9 technology,the iCaspase9 fragment was inserted into the 11^th exon of the DAZL gene to construct Dazl-iCaspase9EGFP PGCs. After identification of iCaspase9 fragment insertion and PGC reproductive characteristics,its ablation effect under B/B homodimerizer induction was identified by fluorescence microscope.【Result】There was no significant difference in growth and proliferation between the successfully established CAG-iCaspase9-mCherry DF-1 cell line and wildtype DF-1 cells(WT DF-1)(P>0.05,the same below). CCK-8 assay revealed that 0.25-5.00 nmol/L B/B homodimerizer had no significant effect on the growth of WT DF-1 cells. However,after adding B/B homodimerizer to CAG-iCaspase9-mCherry DF-1 cells,the number of cells extremely significantly decreased compared to the blank control group(P<0.01, the same below),and normal cell morphology was lost,which gradually becoming round or even apoptotic. The successfully established DAZL-iCaspase9-EGFP PGCs still specifically and highly expressed germ cell related genes such as Cvh,Nanog,PouV,DAZL,Cdh,and Ddx4. Proteins such as DAZL,SSEA1,and CVH could also be identified at the protein level,indicating that the insertion of iCaspase9 fragments did not alter the germ cell specificity of PGCs. Under the induction effect of 0.25 nmol/L B/B homodimerizer,the number of DAZL-iCaspae9-EGFP PGCs significantly decreased and almost all of them were eliminated,indicating that the iCaspase9 system can induce cell apoptosis and effectively eliminate endogenous PGCs.【Conclusion】The DAZL-iCaspase9-EGFP PGCs constructed using the iCaspase9 system can significantly eliminate endogenous PGCs under B/B homodimerizer induction without altering their germ cell characteristics,which provides a receptor chicken embryo without endogenous PGCs for establishing efficient chicken germplasm resource recovery and gene editing chicken preparation research.