In present paper,a study on reconstitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out.Reconstituted oocytes which were the fusion with CC and showed a ...In present paper,a study on reconstitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out.Reconstituted oocytes which were the fusion with CC and showed a cleavage rate of 56.7%,developed into morula (11.7%) and blastocysts (6.7%) phases which were higher than those derived from the fusion with FC( P <0.05).The results of this study also involved the effects of oocyte collection method,activation protocol and maturational age of recipient oocytes during the in vitro develpoment of nuclear transfer embryos which were reconstituted with cultured cumulus cells.The cumulus cells synchronized in G 0/G 1 phases through serum starvation culture,were transferred into enuclated oocytes which were collected by aspiration or dissection method and cultured for 33 or 44 h.Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulsation and 6 DMAP,and cultured for 6 days.As for the oocyte collection methods,activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44 h mature recipients.However,the development rate of reconstituted embryos with 33 h mature recipients were significantly higher( P <0.05) by activation with the combination of electric pulsation and 6 DMAP.These results suggest that the reconstituted porcine embryos derived from cultured cumulus cells can develop into the blastocyst stage and that the development of the former could be improved for the reconstitution with young oocyte cytoplast after the activation with the combination of electric pulsation and 6 DMAP.展开更多
As a part of a basic research project on Xeno-transplantion. we have been engaged in the derivation of embryonic stein cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two ...As a part of a basic research project on Xeno-transplantion. we have been engaged in the derivation of embryonic stein cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 and a 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, marker characterization as well as by in vitro and in vivo differentiation. These porcine EG cells are potentially useful for further basic studies.展开更多
This study was to research the cryopreservation effect of plant antifieeze proteins(AFPs) on day 7 porcine expanded and hatched blastocysts that were frozen in 1.5 M glycerol by conventional slow freezing method. The ...This study was to research the cryopreservation effect of plant antifieeze proteins(AFPs) on day 7 porcine expanded and hatched blastocysts that were frozen in 1.5 M glycerol by conventional slow freezing method. The developmental rates of porcine embryos frozen with 0.5 mg/ml AFPs in freeze medium and cultured for 12 and 24 hours in vitro are 25% and 0%respectively. With the concentration of AFPs increased to 5 mg/ml, the corresponding values became 80% and 40%. The hatched rates for porcine embryos frozen with 0.5 mg/ml and 5 mg/ml of AFPs and cultured for 24 hours in vitro are 0% and 20% respectively..The developmental and hatched rate of the contnrol are all 0%(0/4). These results indicate that the survival rates of porcine expanded and hatched blastocyst can be improved by supplementing freeze medium with plant AFPs.展开更多
To obtain reliable results in quantitative PCR （qPCR） reactions, an endogenous control （EC） gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the e...To obtain reliable results in quantitative PCR （qPCR） reactions, an endogenous control （EC） gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA （rniRNA） genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of rniRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types （P-value〉0.05）. These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm （a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes） and NorrnFinder （an excel plug-in that uses an ANOVA-based model to estimate intra- and inter- group variation to indicate the single most stable EC gene） programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts.展开更多
RNA interference （RNAi） is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus （CSFV） replicatio...RNA interference （RNAi） is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus （CSFV） replication by short hairpin RNA （shRNA） in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon （IFN）-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs （miRNA）, and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNNshRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer （SCNT） embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA- induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.展开更多
Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in derivi...Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.展开更多
文摘In present paper,a study on reconstitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out.Reconstituted oocytes which were the fusion with CC and showed a cleavage rate of 56.7%,developed into morula (11.7%) and blastocysts (6.7%) phases which were higher than those derived from the fusion with FC( P <0.05).The results of this study also involved the effects of oocyte collection method,activation protocol and maturational age of recipient oocytes during the in vitro develpoment of nuclear transfer embryos which were reconstituted with cultured cumulus cells.The cumulus cells synchronized in G 0/G 1 phases through serum starvation culture,were transferred into enuclated oocytes which were collected by aspiration or dissection method and cultured for 33 or 44 h.Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulsation and 6 DMAP,and cultured for 6 days.As for the oocyte collection methods,activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44 h mature recipients.However,the development rate of reconstituted embryos with 33 h mature recipients were significantly higher( P <0.05) by activation with the combination of electric pulsation and 6 DMAP.These results suggest that the reconstituted porcine embryos derived from cultured cumulus cells can develop into the blastocyst stage and that the development of the former could be improved for the reconstitution with young oocyte cytoplast after the activation with the combination of electric pulsation and 6 DMAP.
基金an introductory part of a project supported by National Science Foundation of China(No.39993430).
文摘As a part of a basic research project on Xeno-transplantion. we have been engaged in the derivation of embryonic stein cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 and a 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, marker characterization as well as by in vitro and in vivo differentiation. These porcine EG cells are potentially useful for further basic studies.
文摘This study was to research the cryopreservation effect of plant antifieeze proteins(AFPs) on day 7 porcine expanded and hatched blastocysts that were frozen in 1.5 M glycerol by conventional slow freezing method. The developmental rates of porcine embryos frozen with 0.5 mg/ml AFPs in freeze medium and cultured for 12 and 24 hours in vitro are 25% and 0%respectively. With the concentration of AFPs increased to 5 mg/ml, the corresponding values became 80% and 40%. The hatched rates for porcine embryos frozen with 0.5 mg/ml and 5 mg/ml of AFPs and cultured for 24 hours in vitro are 0% and 20% respectively..The developmental and hatched rate of the contnrol are all 0%(0/4). These results indicate that the survival rates of porcine expanded and hatched blastocyst can be improved by supplementing freeze medium with plant AFPs.
基金supported by the National Basic Research Program of China (Grant Nos. 2006CB102106 and 2007CB947402)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant No. 30621064)
文摘To obtain reliable results in quantitative PCR （qPCR） reactions, an endogenous control （EC） gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA （rniRNA） genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of rniRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types （P-value〉0.05）. These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm （a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes） and NorrnFinder （an excel plug-in that uses an ANOVA-based model to estimate intra- and inter- group variation to indicate the single most stable EC gene） programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts.
基金Project supported by the Major Projects of New Varieties of Genetically Modified Organisms (No.2011ZX08006-001)the Program for Changjiang Scholars and Innovative Research Team in the University (No.IRT1248),China
文摘RNA interference （RNAi） is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus （CSFV） replication by short hairpin RNA （shRNA） in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon （IFN）-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs （miRNA）, and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNNshRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer （SCNT） embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA- induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.
基金supported by the National Basic Research Program of China (Grant Nos. 2009CB941003, 2011CBA0110 and 2011CBA01000)
文摘Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.
