To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the ...To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.展开更多
Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the preve...Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.展开更多
The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine ...The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 mg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blas-tocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, re-spectively). The development rates to the blastocyst stage of NT embryos were significantly dif-ferent among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal ovi-duct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo develop-mental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.展开更多
Adult somatic cell nuclear transfer was con-ducted by using cultured ear fibroblast cells obtained from a Holstein female cow (GN) and a Galoway herd bull (GLV). The percentages of reconstructed eggs developed into bl...Adult somatic cell nuclear transfer was con-ducted by using cultured ear fibroblast cells obtained from a Holstein female cow (GN) and a Galoway herd bull (GLV). The percentages of reconstructed eggs developed into blas-tocysts were similar in GN (23.98%, 123 of 513) and in GLV groups (29.55%, 138 of 467). However, the rate of recon-structed female (GN) embryos developed into term was higher than that of male (GLV) (8.02% and 1.82%, respec-tively). Three kinds of cows, Luxi Yellow cows, Holstein heifers and Holstein cows with normal reproductive records were used as recipients. When the reconstructed embryos from GN were transferred, there was no difference in the pregnancy rate among three kinds of recipients, but the abortion rate of Luxi Yellow cows was significantly higher (85.71%) than in the other two groups (14.29% and 0%, respectively; P < 0.05). And the percentages of newborn calves in transferred embryos were significantly different between Luxi Yellow cows and Holstein breed (1.54%, 10.39% and 20.0%, respectively, P < 0.05). However, when reconstructed embryos from GLV were transferred, there was no difference among three kinds of recipients in the pregnancy rate, the abortion rate and the delivery rate.展开更多
Aquaculture is one of the fastest developing agricultural industries worldwide.One of the most important factors for sustainable aquaculture is the development of high performing culture strains.Genome manipulation of...Aquaculture is one of the fastest developing agricultural industries worldwide.One of the most important factors for sustainable aquaculture is the development of high performing culture strains.Genome manipulation offers a powerful method to achieve rapid and directional breeding in fish.We review the history of fish breeding methods based on classical genome manipulation,including polyploidy breeding and nuclear transfer.Then,we discuss the advances and applications of fish directional breeding based on transgenic technology and recently developed genome editing technologies.These methods offer increased efficiency,precision and predictability in genetic improvement over traditional methods.展开更多
基金supported by grants from the Major State Basic Research Development Program of China(No.001CB5099)the National High Technology Research and Development Program of China(No.2001AA216121)+3 种基金National Natural Science Foundation of China(No.30040003)Projects of Shanghai Science&Technology Development Foundation(No.99DJ14002,00DJ14033,01DJ14003)the Chinese Academy of Sciences(No.KSCX-2-3-08)Shanghai Municipal Education Commission and by Shanghai Second Medical University
文摘To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.
基金suppored by the Na-tional High-Tech Research and Development program (Grant No. 2006AA02Z113)
文摘Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.
文摘The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 mg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blas-tocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, re-spectively). The development rates to the blastocyst stage of NT embryos were significantly dif-ferent among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal ovi-duct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo develop-mental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.
基金supported by the National Natural Science Foundation of China(Grant No.39830280).
文摘Adult somatic cell nuclear transfer was con-ducted by using cultured ear fibroblast cells obtained from a Holstein female cow (GN) and a Galoway herd bull (GLV). The percentages of reconstructed eggs developed into blas-tocysts were similar in GN (23.98%, 123 of 513) and in GLV groups (29.55%, 138 of 467). However, the rate of recon-structed female (GN) embryos developed into term was higher than that of male (GLV) (8.02% and 1.82%, respec-tively). Three kinds of cows, Luxi Yellow cows, Holstein heifers and Holstein cows with normal reproductive records were used as recipients. When the reconstructed embryos from GN were transferred, there was no difference in the pregnancy rate among three kinds of recipients, but the abortion rate of Luxi Yellow cows was significantly higher (85.71%) than in the other two groups (14.29% and 0%, respectively; P < 0.05). And the percentages of newborn calves in transferred embryos were significantly different between Luxi Yellow cows and Holstein breed (1.54%, 10.39% and 20.0%, respectively, P < 0.05). However, when reconstructed embryos from GLV were transferred, there was no difference among three kinds of recipients in the pregnancy rate, the abortion rate and the delivery rate.
基金supported by the National Basic Research Program of China(2010CB126306,2012CB944504)the National Science Fund for Excellent Young Scholars of the National Natural Science Foundation of China(31222052)+1 种基金the Chinese Academy of Sciences Grant KSCX2-EW-N-004-4the State Key Laboratory of Freshwater Ecology and Biotechnology grant 2011FBZ23
文摘Aquaculture is one of the fastest developing agricultural industries worldwide.One of the most important factors for sustainable aquaculture is the development of high performing culture strains.Genome manipulation offers a powerful method to achieve rapid and directional breeding in fish.We review the history of fish breeding methods based on classical genome manipulation,including polyploidy breeding and nuclear transfer.Then,we discuss the advances and applications of fish directional breeding based on transgenic technology and recently developed genome editing technologies.These methods offer increased efficiency,precision and predictability in genetic improvement over traditional methods.
