[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in ...[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation. One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization （IVF）; the other group of oocytes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection （generally called ICSI-Mediated Gene Transfer, ICSI-Tr）. Expression of exogenous DNA was observed and recorded during the process of embryonic development. [Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF injection group showed no significant difference compared with that in ICSI-Tr group （P0.05）. In addition, the cleavage rate and early embryonic gene expression efficiency in IVF injection group were significantly higher with injection at 7-10 h post IVF than that at 18-20 h post IVF （P0.05）. [Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes, and the optimal injection time is 7-10 h post IVF.展开更多
Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig(1876),enormous progress and insights have been made on this topic.However,the precise ...Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig(1876),enormous progress and insights have been made on this topic.However,the precise molecular mechanisms underlying fertilization are largely unknown.The two most dramatic changes taking place in the zygote immediately after fertilization are:(i) a sharp increase of intracellular Ca2+ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave,and(ii) the concomitant dynamic rearrangement of the actin cytoskeleton.Traditionally,this has been studied most extensively in the sea urchin eggs,but another echinoderm,starfish,whose eggs are much bigger and transparent,has facilitated experimental approaches using microinjection and fluorescent imaging methodologies.Thus in starfish,it has been shown that the sperm-induced Ca2+ increase in the fertilized egg can be recapitulated by several Ca2+ -evoking second messengers,namely inositol 1,4,5-trisphosphate(InsP3) ,cyclic ADP-ribose(cADPr) and nicotinic acid adenine dinucleotide phosphate(NAADP) ,which may play distinct roles in the generation and propagation of the Ca2+ waves.Interestingly,it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca2+ signaling.As it is well known that Ca2+ regulates the structure of the actin cytoskeleton,our finding that Ca2+ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca2+ signaling may form a'positive feedback loop'that accelerates the downstream events of fertilization.Perturbation of the cortical actin networks also inhibits cortical granules exocytosis.Polymerizing actin bundles also compose the'acrosome process,'a tubular structure protruding from the head of fertilizing sperm. Hence,actin,which is one of the most strictly conserved proteins in eukaryotes,modulates almost all major aspects of fertilization.展开更多
基金Supported by National High Technology Research and Development (863) Program of China (2011AA100607)National Transgenic Major Project of China (2010ZX08007003)~~
文摘[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation. One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization （IVF）; the other group of oocytes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection （generally called ICSI-Mediated Gene Transfer, ICSI-Tr）. Expression of exogenous DNA was observed and recorded during the process of embryonic development. [Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF injection group showed no significant difference compared with that in ICSI-Tr group （P0.05）. In addition, the cleavage rate and early embryonic gene expression efficiency in IVF injection group were significantly higher with injection at 7-10 h post IVF than that at 18-20 h post IVF （P0.05）. [Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes, and the optimal injection time is 7-10 h post IVF.
文摘Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig(1876),enormous progress and insights have been made on this topic.However,the precise molecular mechanisms underlying fertilization are largely unknown.The two most dramatic changes taking place in the zygote immediately after fertilization are:(i) a sharp increase of intracellular Ca2+ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave,and(ii) the concomitant dynamic rearrangement of the actin cytoskeleton.Traditionally,this has been studied most extensively in the sea urchin eggs,but another echinoderm,starfish,whose eggs are much bigger and transparent,has facilitated experimental approaches using microinjection and fluorescent imaging methodologies.Thus in starfish,it has been shown that the sperm-induced Ca2+ increase in the fertilized egg can be recapitulated by several Ca2+ -evoking second messengers,namely inositol 1,4,5-trisphosphate(InsP3) ,cyclic ADP-ribose(cADPr) and nicotinic acid adenine dinucleotide phosphate(NAADP) ,which may play distinct roles in the generation and propagation of the Ca2+ waves.Interestingly,it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca2+ signaling.As it is well known that Ca2+ regulates the structure of the actin cytoskeleton,our finding that Ca2+ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca2+ signaling may form a'positive feedback loop'that accelerates the downstream events of fertilization.Perturbation of the cortical actin networks also inhibits cortical granules exocytosis.Polymerizing actin bundles also compose the'acrosome process,'a tubular structure protruding from the head of fertilizing sperm. Hence,actin,which is one of the most strictly conserved proteins in eukaryotes,modulates almost all major aspects of fertilization.
