Tracking The Migration And Differentiation Of Chicken Germ Cells By Dazl Fluorescent Reporter

The Induced Pluripotent Stem Cells(iPSC)have the potential to differentiate into multiple cell types.In particular,iPSC differentiation into germ cells in vitro is of great significance for use in animal genetic and transgenic research and developmental biology research.Dazl is an important germ cell marker gene responsible for regulating the germ cell development and differentiation,which has been used as a marker for characterization of germ cells.As Dazl is not expressed in iPSC,it could be used as a reporter to track iPSC differentiation into germ cells.In this study,CRISPR/Cas9(Clustered regular interspaced short palindromic repeats,CRISPR)mediated Homology-Mediated End Joining(HMEJ)was used to insert mCherry into the 10th intron of chicken endogenous Dazl gene.Expression of mCherry driven by endogenous Dazl promoter could be used as reporter to tack the process of iPSC differentiation into PGC.The results the study are as follows:(1)Identification of DAZL expression in chicken PGC and iPSC.First,chicken Dazl gene was cloned and DAZL polyclonal antibody was prepared in rabbit serum after inoculation.Expression of DAZL in PGC was confirmed by Western blot and a specific band with a relative molecular weight of 33 KDa was detected,consistent with the predicted molecular weight.It indicated the specificity of DAZL antibody.q-PCR and immunofluorescence staining results showed that DAZL was not expressed in iPSC,but highly expressed in PGC.These results suggested that Dazl could be used as a specific reporter gene to track germ cells.(2)Targeting integration of Dazl fluorescence reporter.In order to insert mCherry fluorescent reporter gene into Dazl gene by CRISPR/Cas9,we constructed hp180-sgRNA-spCas9 vector and pDazl-HMEJ donor reporter vector.DF-1 cells were transfected with hp180-sgRNA-spCas9 to verify the targeting efficiently.T7E1 analysis revealed that this plasmid could effectively target gene of interest for gene knockout.Then hp180-sgRNA-spCas9 expression vector and pDazl-HMEJ donor vector were co-transfected into PGC and iPSC.PGC cell lines carrying mCherry+/EGFP+and iPSC cell lines carrying mCherry/EGFP+ were successfully established.(3)Tracking of PGC migration and iPSC differentiation by Dazl fluorescence reporter.PGC carrying mCherry+/EGFP+ were injected into the blood system of 15HH chicken embryos.The cells were found to migrate and home to the embryonic gonads 5 days after transplantation.In addition,mCherry+/EGFP+ cells were also found in the testis of newborn chickens,confirming the efficacy of the Dazl fluorescence reporter in tracking the migration of germ cells.Finally,we tracked the differentiation of chicken iPSC into germ cells through embryoid body formation and found EGFP and mCherry double positive cells for 3 days after culture of EB in differentiation medium,in addition EGFP and mCherry double positive cells up-regulated germ cell specific genes and down-regulated pluripotent related genes.The results suggested that Dazl fluorescence reporter could be used as an indicator of differentiation of iPSC into germ cells in vitro.In summary,The Dazl fluorescence reporter system was established by CRISPR/Cas9 technology in this study and could be used in tracking of the migration of PGC and differentiation of germ cells from iPSC in vitro.Results of the research would facilitate the derivation of germ cells and use of these cells in genetic conservation of endangered birds and transgenic research as well as in developmental biology.