Studies On The Functional And Regulatory Network Of DAZL Gene In Chicken Primordial Germ Cells

DAZL(Deleted in azoospermia-like)is a germ cell specific gene,showing higher expression in primordial germ cells(PGCs).In mammalian species,DAZL regulates the potential of PGCs/pluripotent stem cells to self-renew and differentiate by binding the RNA of downstream genes.The researchers have been exploring the role of DAZL in various mammalian species.However,very few studies are available to demonsrate its function in maintenance of chicken germline characteristics.A comprehensive understanding of DAZL gene functions in PGCs could help to elucidate the development and differentiation processes of germ cell lines in avian species.Furthermore,it could provide resources to improve the developmental potential PGCs in vitro,optimize the culture system,and ultimately enhance the efficiency of transgenic chicken production.Here,we studied the DAZL gene functions in chicken PGCs using CRISPR/Cas9 system and omics tools.In the present study,we have established a stable and highly efficient genetic engineering system for chicken.This genetic engineering system offers a promising reference for future studies in transgenic chicken production.The brief description of the present study is as below.1.The application and optimization of CRISPR/Cas9 system in chicken gene editingIn the present study,we confirmed the efficiency of CRISPR/Cas9 system by using the truncated EYFP reporter plasmid and applying this system for editing the endogenous DAZL gene.To enhance the gene knock out efficiency of CRISPR/Cas9 system,we used multi-target sites strategy on MSTN gene in chicken DF-1 cells.In the gene knock out results,we observed the high efficiency of multi-targets methods in chicken cells and the fragments elimination in targeted sites.In order to increase the gene knock in efficiency of CRSIPR/Cas9 system,we compared HITI(Homology-independent targeted integration),HDR(Homology-directed DNA repair)and HMEJ(Homology-mediated end joining)method.And we found that the HMEJ is most consistent and efficient method for gene knock-in in chicken cells.2.DAZL gene silencing and it function in PGC proliferationThe strategy of gene silencing was used to elucidate the DAZL gene functions.At first,we knocked out DAZL gene in PGCs by using multi-targets method and found a significant reduction in the PGCs proliferation.For better tracking this process,we chose HMEJ strategy to insert m Cherry gene into the6 th exon of DAZL to silence the gene.After the insertion of m Cherry(DAZL gene silence),we observed hampered proliferation of PGCs.These results suggested the essential role of DAZL in chicken PGCs survival and proliferation.To further confirm the role DAZL in PGC proliferation,we used Cas RX method for DAZL gene silence at RNA level.After gene silencing mediated by Cas RX,we found the similar retarded proliferation in the PGCs as in the DAZL kockout ones.3.Analysis of regulateory network of DAZLTo better understand the gene regulatory networks of DAZL in PGCs,we used the micro-proteomics to analyze the DAZL-silenced PGCs and explored the related gene regulated network of DAZL.To determine the directly regulating RNA and proteins,we used RIP-Seq(RNA immunoprecipitation-Sequencing)and Co-IP/MS/MS(Co-Immunoprecipi-tation-Liquid Chromatography/Magnetic Deflection/Magnetic Deflection)to analze the DAZL-binding molecues.Analysis of the data revealed the involvement of DAZL in maintenance of cell proliferation,pluripotency and germ-line characteristic in chicken PGCs.In addition,we analyzed the gene regulatory network of CVH,an important downstream gene of DAZL,and found it is part of closely regulatory genes of DAZL in chicken PGCs as seen in other species.4.Production of transgenic chicken with fluorescence labeled DAZLTo facilitate the tracking of PGC and research of original,migration and differentiation process,we tried to produce chickens with DAZL labeled fluorescent gene.Firstly,we established an m KO-FI culture system based on several culture systems for PGC.Next,we optimized transfection methods in fresh deriving PGCs and successfully performed the plasmid DNA transfection into PGCs.Then we generated the DAZL-2A-m Cherry labeled PGCs lines in via HMEJ knock-in method.After the injection of modified PGCs into recipient chicken embryo,we found the significantly migration and colonalization of PGCs in the testis the founder chicks.In general,this research expands our knowledge of function and gene regulatory network of DAZL in chicken PGCs.The genetic engineering and omics analysis strategies established in this research would also provide a resource to elucidate the gene function and be helpful for transgenic chicken production in the future.