Study On Male Germ Cell Derived From Chicken Embryonic Stem Cell And The Generation Of Transgenic Chicken

In the sexual reproduction of organisms, germ cells undertake the genetic information passed in generations and generate new individuals by sperm-egg binding. The characteristic of the germ cells make it become one of the research objects of animal embryology, developmental biology and cell biology. Male germ cells could be used to replace embryonic stem cells for disease therapy and genetic modification since it has the merits of pluripotency and do not involve in ethical and immune rejection problem, therefore, it caused great concern in the biological community. It’s necessary and urgent to begin the research of how to induce stem cells from other tissues differentiated into spermatogonial stem cells. Recently, the research on ESCs differentiated into male germ cells has already been done. But the efficiency is still low.Poultry embryonic stem cells has no restricted within ethics as to human embryonic stem cells(hES) and mouse embryonic stem cells(mES), and can easily be obtained, holding an dominant advantage in simulation clinical treatment. Hence, in this study, poultry species were used as animal model in this study. On the basis of the difference between ES and SSCs, retinoic acid(RA) concentration wes initially screened, in combination with three matrix proteins, sertoli cells, testicular abstracts and testicular cell conditioned medium obtained from different periods, to identify the efficiency of germ cell differentiation derived from chicken ESCs. The study provides conference for build appropriate induction system and clinical research. Meanwhile used in vivo SSCs-mediated way transfer active-antiviral MMx protein to generate antiviral transgenic chicken, and provided new ways and thinking for prepared transgenic animals and cultivated new species. The results addressed as follows:(1)To identify the two stem cells, the similarities and differences between the chicken embryonic stem cell (ESC) and spermatogonial stem cell (SSCs) were studied. The characteristics of ESC and SSCs were analyzed with the expression of alkaline phosphatase (AKP), immunologic markers and pluripotency markers. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence and western blotting were used on the two cells. Of the ten genes we detected, Oct4was detected in both of the two cells, while GDF3、Nanog、 Sox2genes only expressed in undifferentiated ES and Dazl, Cvh, Stra8, integrina6, integrinβ1and C-kit genes were found in SSCs, which showed there are differences in ES and SSCs at the level of mRNA. Immunofluorescence and western blotting showed that Sox2protein expressed in ES while C-kit, integrina6, integrinβ1and Dazl proteins were found in SSCs. Our result showed chicken ES and SSCs had common characteristics of stem cell, but exhibited significant discrepancy at the level of mRNA and protein. The different specific markers between the two cells not only suggested the differentiation potential of the two cells but also offered theoretical and practical basis for identifying the two cells.(2) In this part, On the basis of identifying the chicken ES cells sex, the ZZ ES cells were transferred onto24-well plates. For screening the suitable concentration of RA, both cell morphologic and genes expression were detected under different concentration (10-3mol/L、10-4mol/L、10-5mol/L、10-6mol/L). The results showed10-5mol/L was the best. Under this concentration, embryonic body was observed on induced4days. With the prolongation of induction time, EB became large gradually. Until induced8d, EB began to disintegrate and became single round cells. SSCs-like cells did not be observed on the induced10days. On this induction process, expression of many genes changed gradually. ES specific genes Nanog and Sox2expression descended while germ cell specific genes Stra8、Dazl、integrina6、intrgrinβ1、C-kit ascended especially Stra8. we supposed RA was in charge of notable heighten of Stra8expression. In conclusion, RA can promote ES differentiation into germ cell.(3)ES cells developed in the specific niche. The proliferation and differentiation of ES cells are affected by the interaction between variety of matrix proteins and other cells. This experiment studied the possibility of ES cells differentiated to germ cells when ES directly contacted with collagen, fibronectin and laminin under the RA medium. The results showed:on the RA+fibronectin group, embryonic body was observed at induced4days while on the other two groups were induced6days. On RA+fibronectin and RA+laminin groups, SSCs-like cells were seen on induced10days while RA+collagen group did not been observed on induced10days. Related gene expression changes were more significantly. The expression of Nanog and Sox2genes gradually descended especially RA+fibronectin group. We found distinctive difference on Dazl expression between the three groups. On the RA+collagen group, the expression of Dazl gene rising from induced2days, and reached the peak at8days, then descended on10days. On RA+laminin group, Dazl expression reached the peak at induced4days, then gradually descent. On RA+fibronectin group, the expression of Dazl gene began to express from the2st day and expressed significantly at the4th day, then maintained a higher level at the6th,8th and10th. The expression of Stra8、integrinβ1、integrina6genes increased as induced time prolonging. Conclusion:as the induced time prolonging, the expression of Nanog and Sox2genes descent and C-kit, Stra8, integrin(31, integrina6ascend. Compared on cell morphologic change and specific genes expression, the RA+marix protein groups had better induce effection than RA especially RA+fibronectin.(4) Research suggested sertoli cells are the key factor on SSCs niche, which can secrete some growth factors to regulate the progress of germ cell development. In this experiment, different methods were used to induce ES differentiation into germ cell, which were testes abstract and testes medium from different periods, chichen embryonic sertoli cells co-culture with ES and comprehend factors.The results were:On the chicken embryonic sertoli cells group, embryonic body was observed on the induced2days and SSCs-like cells on the induced10days less. Nanog gene expressed lower as time going induced by RA. The expression of Dazl was seen at the peak on induced2days and gradually descent following. Stra8, integrin(31and integrina6genes began to express from the2st day and expressed significantly at the4th day, then maintained a higher level at the6th,8th and10th. On the groups of RA+testes abstract and testes medium from different periods, embryoid was observed on the induced4days and SSCs-like cells on the8d at sexual maturity testes abstract and testes medium groups while on the10days on the other groups. Nanog gene expressed higher as time going induced by RA while Sox2is lower. Dazl、Stra8、integrinβ1、and integrina6expression were gradually ascend. On the comprehend group, embryoid was seen on induced2days and SSCs-like cells on the8d. Nanog and Sox2genes expressed lower than RA group as time going and Dazl, Stra8, integrinp1, integrina6and C-kit higher. Conclusion:under RA+testes abstract and testes medium from different periods, sexual maturity testes abstract and testes medium induced effect was better than the other groups. The induction effect of comprehend group (RA+fibronectin+sertoli cells) was almost same as the sertoli cells group. Between those groups, RA+sertoli group is the best induced methods.(5)In order to generate anti-viral transgenic chickens through transfected spermatogonial stem cell with fusion gene EGFP-MMx. After injecting fusion gene EGFP-MMx into testes, tissues frozen section, PCR and dot blot of testes was performed at the30d,40d,50d,60d,70d and80d, a few of normal hen were inseminated artificially with cock semen of60d. Finally PCR, reverse transcription-PCR and Western Blot were used to detect the integration of exogenous gene EGFP-MMx of the offspring. Tissues frozen section results showed18experimental chickens have the green fluorescence and the number and brightness of fluorescence was at the highest level. DNA was extracted from18experimental chicken and PCR and dot blot were preformed to detect the EGFP and MMx gene. The positive rate was77.8%and72.2%respectively. The four experimental chickens’ sperm were collected and DNA was abstracted for PCR performing. The PCR positive rate was25%(1/4). PCR and RT-PCR positive rate was10.53%(438) in F1blood genome and RNA respectively, while Western Blot positive rate of F1blood was7.89%(3/38). Taken together, it was proved the possibility of cultivating anti-viral transgenic chickens by the method of testes injection method which could integrate foreign genes into the genomeIn conclusion, we compared different methods effect on chicken ES differentiation to male germ cell and explored the possibility of cultivating anti-viral transgenic chickens by the method of testes injection method:under the RA medium, the group of ES cells cocultured with sertoli cells group has the best effect on chicken ES differentiation to male germ cell; by SSCs-mediated in vivo, we obtained3transgenic chickens with antiviral gene. The results can provide the theoretical basis for SSCs clinical application and provide an idea for breeding new variety...