To explore the pluripotency maintenance and update the functional influence of pluripotency genes cNanog and cPouV in chicken ( C,a/lus gallus) embry- onic stem cells ( cESCs), the stable RNAi vectors pSuper-cNano...To explore the pluripotency maintenance and update the functional influence of pluripotency genes cNanog and cPouV in chicken ( C,a/lus gallus) embry- onic stem cells ( cESCs), the stable RNAi vectors pSuper-cNanog and pSuper-cPouV constructed previously were used to transfect cESCs. The mRNA levels of two target genes were detected with real- time PCR. These two genes were down-regulated since the 48^th and the down-reg-lation continued with the extension of time, the interference efficiency reached 65% at 96^th hour (P 〈0.05). With the down-regulation of cNanog or cPouV gene, cESCs showed differentiation and prolifera- tion rate of these cells slowed down, the domed colony of these cells disappeared gradually when the edge of colony became irregular. At 96^th hour after transfection, the alkline phosphatase (AKP) and stage-specific embryonic antigen-1 ( SSEA-1 ) were not be detected in cNanog gene-knecked out eESCs, but it was done in that with cPouV gene -knocked out. The cPouV-suppressing cESCs were again transfected with pSuper-cNanog, the pluripotency markers AKP and SSEA-1 were both not found expressing at the 48^th hour. The results showed that cPouV and cNartog genes played an important role in maintaining pluripotency and self- renewal in cESCs, and cNanog gene was dominant. To sum up, our results may provide insights into the molecular regulation mechanism of avian during development.展开更多
Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro....Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.展开更多
Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro a...Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.展开更多
基金Supported by National Natural Science Foundation of China(No.31072101No.31201871)Natural Science Foundation of Guangdong Province
文摘To explore the pluripotency maintenance and update the functional influence of pluripotency genes cNanog and cPouV in chicken ( C,a/lus gallus) embry- onic stem cells ( cESCs), the stable RNAi vectors pSuper-cNanog and pSuper-cPouV constructed previously were used to transfect cESCs. The mRNA levels of two target genes were detected with real- time PCR. These two genes were down-regulated since the 48^th and the down-reg-lation continued with the extension of time, the interference efficiency reached 65% at 96^th hour (P 〈0.05). With the down-regulation of cNanog or cPouV gene, cESCs showed differentiation and prolifera- tion rate of these cells slowed down, the domed colony of these cells disappeared gradually when the edge of colony became irregular. At 96^th hour after transfection, the alkline phosphatase (AKP) and stage-specific embryonic antigen-1 ( SSEA-1 ) were not be detected in cNanog gene-knecked out eESCs, but it was done in that with cPouV gene -knocked out. The cPouV-suppressing cESCs were again transfected with pSuper-cNanog, the pluripotency markers AKP and SSEA-1 were both not found expressing at the 48^th hour. The results showed that cPouV and cNartog genes played an important role in maintaining pluripotency and self- renewal in cESCs, and cNanog gene was dominant. To sum up, our results may provide insights into the molecular regulation mechanism of avian during development.
文摘Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.
基金supported by MOST National Major Basic Research Program(Grant Nos.2010CB94500,2009CB941000(to LL),and 2010CB833703(to FY)).
文摘Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.