The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell ...The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.展开更多
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
Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole)...Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and electroactivation.According to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:1.thosewith chromosomes juxtaposed to the polar body;2.those with chromosomes in its adjacency; and3.those with chromosomes further removed.The relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further on.In addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 hours.As aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 hours.By so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.展开更多
基金This work was supported by the National High Technology Research and Development Program of China(863 Program)(No.2001 AA213081).
文摘The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.
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.
文摘Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and electroactivation.According to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:1.thosewith chromosomes juxtaposed to the polar body;2.those with chromosomes in its adjacency; and3.those with chromosomes further removed.The relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further on.In addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 hours.As aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 hours.By so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.
