[Objective] The research aimed to lay the foundation for producing the transgenic clone pig.[Method] The pig fetus fibroblast with the red fluorescent protein（RFP）gene that was transfected by the retrovirus was as t...[Objective] The research aimed to lay the foundation for producing the transgenic clone pig.[Method] The pig fetus fibroblast with the red fluorescent protein（RFP）gene that was transfected by the retrovirus was as the donor of nucleus transplantation.By using the somatic cell cloning technology,the development situation in vitro of clone embryo with RFP was studied.[Result] The fusion rate of RFP transgenic cell was 83.87% which had no significant difference with 80.56% of non-transgenic cell（P0.05）.The blastula rate in vitro of RFP transgenic somatic cell reconstructed embryo was 8.67% which had no significant difference with 6.56% of non-transgenic cell（P0.05）.After the reconstructed embryo of RFP transgenic somatic cell was transplanted into fifteen receptors,there was no conception individual.[Conclusion] The transgenic cell with the red fluorescent protein as the donor could successfully clone the transgenic embryo and obtain the transgenic blastula.展开更多
Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been th...Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century展开更多
This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (St...This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.展开更多
Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has be...Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.展开更多
Research in the field of somatic cell nuclear transfer （SCNT） and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotranspl...Research in the field of somatic cell nuclear transfer （SCNT） and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low （ 〈 1% ）. Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.展开更多
基金Supported by the National Natural Science Foundation Item(30960175)"New Variety Cultivation Key Special Item of Transgene Organisms" of Ministry of Agriculture(2009ZX08006-002B)~~
文摘[Objective] The research aimed to lay the foundation for producing the transgenic clone pig.[Method] The pig fetus fibroblast with the red fluorescent protein（RFP）gene that was transfected by the retrovirus was as the donor of nucleus transplantation.By using the somatic cell cloning technology,the development situation in vitro of clone embryo with RFP was studied.[Result] The fusion rate of RFP transgenic cell was 83.87% which had no significant difference with 80.56% of non-transgenic cell（P0.05）.The blastula rate in vitro of RFP transgenic somatic cell reconstructed embryo was 8.67% which had no significant difference with 6.56% of non-transgenic cell（P0.05）.After the reconstructed embryo of RFP transgenic somatic cell was transplanted into fifteen receptors,there was no conception individual.[Conclusion] The transgenic cell with the red fluorescent protein as the donor could successfully clone the transgenic embryo and obtain the transgenic blastula.
文摘Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century
基金Project (No. R-174-000-065-112/303) supported by the NationalUniversity of Singapore
文摘This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.
基金supported by grants from Ministry of Science and Technology of China(2011CBA01001,2012AA020503)the National Science Fund for Distinguished Young Scholars(31025016)+4 种基金the National Natural Science Foundation of China(31271577)Novel Agricultural Variety Breeding Project of Zhejiang Province(2012C12906-8)the Fundamental Research Funds for the Central Universitiesthe Key Construction Pro-gram of the National"985"Project(118000+193411801/006)the Research Fund for the Doctoral Program of Higher Education of China(20120101110089)
文摘Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.
文摘Research in the field of somatic cell nuclear transfer （SCNT） and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low （ 〈 1% ）. Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.
