A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst a...A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g010 was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.展开更多
Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supp...Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonqui-escent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blas-tomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial Ⅱ and serial Ⅲ were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups展开更多
Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody againstubiqui...Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody againstubiquitinated proteins——UCPi; the polyclonal antibodyagainst ubiquitin-anti-Ub, and a special inhibitor againstproteasome——ALLN. Furthermore, we treated the capaci-tated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction, but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.展开更多
Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogen-activated protein kinase (MAPK) was detected with SDS-PA...Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogen-activated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK2 was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphory-lation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while展开更多
The nucleus of a somatic cell could be dedif-ferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term no...The nucleus of a somatic cell could be dedif-ferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellu-cida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic展开更多
文摘A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g010 was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.
基金This work was supported by the Climbing Project of the Ministry of Sciences and Technology of China (Grant No.97021109-2)the Important Project of Knowledge Innovation of Chinese Academy of Sciences (Grant No. KSCX1-05-01).
文摘Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonqui-escent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blas-tomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial Ⅱ and serial Ⅲ were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups
基金This work was supported by the Special Funds for Major State Basic Research Projects (Grant No. G1999053901) the National Natural Science Foundation of China (Grant No. 39430080).
文摘Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody againstubiquitinated proteins——UCPi; the polyclonal antibodyagainst ubiquitin-anti-Ub, and a special inhibitor againstproteasome——ALLN. Furthermore, we treated the capaci-tated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction, but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.
基金This work was supported by the Major State Basic Research ("973") Project (Grant No. G1999055902)the Special Project of Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-SW-303).
文摘Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogen-activated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK2 was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphory-lation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while
基金This work was supported by the fund of Climbing Program of the Ministry of Science and Technology (Grant No. 95-specific-08) and the Chinese Academy of Sciences (Grant No. KSCX1-05-01).
文摘The nucleus of a somatic cell could be dedif-ferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellu-cida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic
