Establishment The System Of Chicken CEF Reprogramming Induce To PGC And Research Of Its Migration Homing Fuction

The process of improved livestock production since the 19th century has made the global livestock breeds gradually simplistic,and a large number of genetic resources,especially local varieties,have been destroyed.Until the beginning of the 21st century,45%of the 7616 livestock species known worldwide were on the verge of extinction.In mammalian livestock,somatic cell nuclear transfer,in vitro fertilization and embryo transfer techniques have been successful,combined with the cryopreservation of sperm,eggs,embryos and even somatic cells to achieve germplasm conservation and species restoration.Due to the characteristics of the oviparity,the above methods are difficult to achieve on poultry.The primordial germ cell(PGC)of poultry has a unique feature of colonization by blood migration,making the preparation of reproductive chimeras by PGC the only way to achieve the recovery of avian species.However,the PGC acquired by a single embryo is far from enough to meet the needs of practical applications.In particular,the source of PGCs for endangered species has become a problem limiting this technology.While somatic cells can be obtained in large quantities in a short period of time,if they can be induced into PGCs,it is the best way to solve this difficulty.In order to solve the problem of chicken cell induced PGC,this study firstly established the chicken embryonic fibroblast(CEF)of Langshan chicken by single cell cloning technology,and obtained Chicken embryonic fibroblast single cloned cell line-Langshan,CSC-LS cell line.In CEF and CSC-LS,the lentiviral overexpression of Oct4,Sox2,Nanog and Lin28 binds to the BMP4/BMP8b/EGF induction system,and CEF is reprogrammed to induce iPGC with the ability to form reproductive chimerism.The black-feathered pheasant was used as a donor,and the recessive white-feathered chicken was used as a receptor.A method for the production of immature chimeric chicken by iPGC induced from CEF was established.At the same time,the progeny of donor PGC was produced by preparing PGC reproductive chimeras of black feather pheasant and recessive white chicken,which verified the feasibility of PGC pathway and laid the research foundation for the application of iPGC.The results summarize as follows:(1)Establishment of CSC-LS CEF single cloned cell line.Continuous subculture of Wolf Mountain chicken CEF found that continuous subculture of CEF will undergo apoptosis and enter a proliferative crisis.The CSC-LS cell line establish by single cell isolation and subculture.Optimization of the culture system of single-cell clones found that Ham's F2 is the optimal medium for the preparation of single-cell clones with a cloning efficiency of 25%±0.01,which is significantly higher than DMEM(11%±0.01)and D/Fl2 Medium(12%±0).The efficiency of monoclonal preparation of the mouth pipette method was 32.29%±0.04 significantly higher than the 6.95%± 0.01 of the limiting dilution method.The CSC-LS cell line had a normal diploid karyotype compared with normal CEF,but its proliferative capacity was significantly enhanced(P<0.01),and the growth curve was inverted S-type.The proportion of cell cycle G2/M phase was 29.3±0.01 significantly higher than the original CEF 19.12%± 0.01.The expression levels of proto-oncogenes Ras,c-myc,and Src in CSC-LS were 0.95±1.01,2.70±0.21 and 1.27±0.06,respectively,which were significantly lower than 0.97±0.99,4.95±0.50 and 11.84±1.27 of DF-1(P<0.05),significantly lower than 13.49±2.7,19.21 ± 1.23,16.58±1.53(P<0.01)of DT40 cells.CSC-LS increased the transfection efficiency of liposome transfection plasmid vector,and the transfection efficiency was 51.3%±0.01 significantly higher than 1.3%±0.003 of primary CEF(P<0.01).(2)Reprogramming induction from CEF to iPGC.Oct4,Sox2,Nanog,and Lin28 lentiviral overexpression vectors were constructed.The infection conditions of lentivirus for CEF and CSC-LS were optimized,and 5 jig/ml polybrene with MOI=10 was determined as the best infection condition.During the CEF reprogramming process,the cell morphology was over-expressed by the lentivirus over the reprogramming factor.On the 5th day,the fibroblasts gradually became rounded and aggregated to form a clone.On the 15th day,the morphology of the iPS cells gradually became clear,and after 17 days,the formation of clear cells began to form.Clonal colonies were able to subculture and reconstitute cell clones by monoclonal selection.The OSNL-mediated iPS induction efficiency was 0.59%±0.03.On the basis of this,the iPS induction efficiency after adding VPA was 1.36%±0.24,VPA+VC was 1.66%±0.01,which was significantly higher than that of the group that only overexpressed OSNL(P<0.01).It was preliminarily determined that OSNL+VPA+VC was the best iPS induction system and subsequently identified.The obtained iPSC can be labeled with SSEA-1,stained with alkaline phosphatase,and has a diploid karyotype Compared with CEF,the pluripotency genes Nanog,Oct,Sox2,SSEA-1,CLCD3,Klf4,Rps17,SALL4 were highly significant(P<0.01).Edu staining showed that 53.30%±0.23 cells in iPSC were significantly proliferating compared with 6.40%±0.05 of feeder layer CEF(P<0.01).The expression changes of endogenous pluripotency genes of CEF and CSC-LS were detected from day 3 post-infection to 21 days after infection.It was found that CEF can activate endogenous pluripotency genes Oct4,Sox2,Nanog,Lin28 and SSEA-1 during reprogramming.The expression levels of Oct4,Sox2,and Nanog reached the highest on the 21st day,which were 12.95±1.7,132.1±7.7,and 22.21±2.3,respectively.The expression level of Lin28 on day 15 was 25.11±3.08,and CSC-LS could not activate the expression of Sox2,SSEA-1 and Oct4.The DNA methylation level of promoters of Oct4,Sox2,Nanog and Lin28 showed that the DNA methylation levels of Oct4,Sox2 and Nanog continued to decrease during the induction process,but there was no significant change in Lin28.Based on the previous establishment of ESC to PGC induction model in vitro optimization experiments,the results showed that compared with BMP4 induction process,BMP8b and EGF can increase the induction efficiency of ESC to PGC to 26.2%±0.01 compared with 7.3%±0.01 of BMP4 group.The difference was extremely significant(P<0.01).iPSCs were induced to iPGC by the same system,cells began to aggregate to form embryo bodies at 2 days of induction,and gradually aggregated and increased after 4 days.The induction efficiency of iPGC reach a maximum of 12.2%±0.01 on the fourth day;the obtained iPGC expressed Cvh protein;the glycogen particles in the iPGC could be stained by Schiff reagent;the iPGC can express the marker gene of PGC Cvh,C-kit,Blimpl and part of iPSC pluripotency genes Nanog,Sox2,Oct4.iPGC was injected into the chicken embryo in 2.5 hatching days.It was observed in real time that iPGC could migrate to the reproductive ridge through the blood,and its green fluorescence could be observed by frozen section,and the expression of GFP could be detected by Western blotting.Epigenetic modification was involved in the regulation of the whole induction process:histone H3K4me2 increased first and then decreased during the induction of CEF to iPGC,and decreased gradually after iPSC induced the highest level on iPGC on the fourth day.The histone acetylation level was in CEF.The induction process to iPSC showed an upward trend,and iPSC increased to the iPGC during the induction process on the second day(3)Establishment of PGC germline chimeric chicken model.The PGC isolated from the reproductive ridge is purified by feeder-free culture.the glycogen particles in the purified PGC can be stained with Schiff reagent.Cvh and C-kit proteins can be detected in PGC.The expression levels of PGC marker genes c-kit,Blimp1,Dazl and Cvh were 17.91±0.43,56.26±3.60,14.73±0.23,and 82.22±2.63,respectively,which were significantly different from CEF(P<0.01).The gonad-directed migration ability of CEF,ESC,and PGC,iPGC was analyzed by chicken embryo blood vessel injection test.It was found that only PGC and iPGC could migrate to the reproductive ridge to form a reproductive chimera by intravascular injection.PGC germline chimeric chickens were obtained by 2.5-day PGC vascular injection.Germline chimeras were detected by frozen sections in 18.5 days of chicken embryo gonads.By injecting PGC of wolf pheasant and recessive white feather into the chicken embryo of each other,it can form germline chimeras and produce offspring of PGC.The four experimental groups are normal white hen x experimental black cock,experimental black hen xNormal white cock,experimental white hen x normal white cock,normal white hen x experimental white cock.F2 generation chicken were detected by the microsatellite locus LEI094,the length of LEI094 is 290,294,recessive white chicken LEI094 The length was 311,and the germline chimeric efficiency was 12.04%,10.11%,8.96%,8.70%,and the average chimeric efficiency was 9.95%.