Method for producing parthenogenetic cloned pig by means of continuous cloning technology

Abstract

The invention relates to a method for producing a parthenogenetic cloned pig by means of continuous cloning technology, which belongs to the field of reproductive biology. The method provided by the invention comprises the following steps of: the degreasing of a porcine oocyte, parthenogenetic activation, the separation of a fibroblast, and continuous somatic nuclear transfer; and specifically, the method comprises the following steps of: carrying out the parthenogenetic activation on the degreased mature porcine oocyte to obtain a parthenogenetic embryo, transplanting the parthenogenetic embryo into the body of a surrogate sow to obtain a 25-29 days old living parthenogenetic fetus, establishing a porcine parthenogenetic fetus fibroblast line which is used as a preliminary donor cell, and carrying out continuous cloning by means of somatic cell nuclear transfer technique to obtain a porcine parthenogenetic individual. By adopting the method provided by the invention, the condition that the in-vivo development stops when the porcine parthenogenetic fetus grows for 30 days is overcome, the in-vivo development time of the porcine parthenogenetic fetus is successfully prolonged, and therefore a living parthenogenetic cloned pig can be successfully obtained finally.

Description

technical field

[0001] The invention relates to a method for producing parthenogenetic cloned pigs by using continuous cloning technology, using parthenogenetic fetal fibroblasts as initial donor cells, and using somatic cell nuclear transfer technology for continuous cloning to obtain surviving parthenogenetic pig individuals , belonging to the field of reproductive biology. Background technique

[0002] Parthenogenesis is the process of asexual reproduction in which an embryo is produced by a female gamete without any action by the male gamete, whether or not it occurs into an individual. Parthenogenesis has important application value for elucidating the signal coupling process of mammalian egg activation process, recovery of cell cycle arrest, fertilization mechanism and developmental mechanism. In addition, due to the absence of paternal genes, parthenogenesis is of great significance to the study of mammalian embryos, placental growth and imprinted genes. Haploid and...

Examples

Embodiment 1

[0031] Using somatic cell nuclear transfer technology to continuously clone pig fetal fibroblasts, and finally obtain surviving pig individuals, the specific implementation methods are as follows:

[0032] The first step is to obtain pig parthenogenetic fetal fibroblasts;

[0033] The pig ovaries are collected from the slaughterhouse, stored in normal saline and transported back to the laboratory, where the pig oocytes are picked and matured in vitro. The mature oocytes are centrifuged at high speed (13000rmp, 20min), so that the fat particles are aggregated into clusters and the fat particles are removed by micromanipulation technology. Then parthenogenetic activation is performed on it to obtain parthenogenetic embryos, and the parthenogenetic embryos are transplanted into surrogate sows that are in estrus on the same day to continue to develop.

[0034] On the 23rd day, the sows were diagnosed with pregnancy, and the 25-day-old surrogate sows were subjected to laparotomy t...

Embodiment 2

[0042] Using somatic cell nuclear transfer technology to continuously clone pig fetal fibroblasts, and finally obtain surviving pig individuals, the specific implementation methods are as follows:

[0043] The first step is to obtain pig parthenogenetic fetal fibroblasts;

[0044] The pig ovaries are collected from the slaughterhouse, stored in normal saline and transported back to the laboratory, where the pig oocytes are picked and matured in vitro. The mature oocytes are centrifuged at high speed (13000rmp, 20min), so that the fat particles are aggregated into clusters and the fat particles are removed by micromanipulation technology. Then parthenogenetic activation is performed on it to obtain parthenogenetic embryos, and the parthenogenetic embryos are transplanted into surrogate sows that are in estrus on the same day to continue to develop.

[0045] On the 23rd day, the sows were diagnosed with pregnancy, and the 25-day-old surrogate sows were subjected to laparotomy t...

technical field

[0001] The invention relates to a method for producing parthenogenetic cloned pigs by using continuous cloning technology, using parthenogenetic fetal fibroblasts as initial donor cells, and using somatic cell nuclear transfer technology for continuous cloning to obtain surviving parthenogenetic pig individuals , belonging to the field of reproductive biology. Background technique

[0002] Parthenogenesis is the process of asexual reproduction in which an embryo is produced by a female gamete without any action by the male gamete, whether or not it occurs into an individual. Parthenogenesis has important application value for elucidating the signal coupling process of mammalian egg activation process, recovery of cell cycle arrest, fertilization mechanism and developmental mechanism. In addition, due to the absence of paternal genes, parthenogenesis is of great significance to the study of mammalian embryos, placental growth and imprinted genes. Haploid and...

Examples

Embodiment 1

[0031] Using somatic cell nuclear transfer technology to continuously clone pig fetal fibroblasts, and finally obtain surviving pig individuals, the specific implementation methods are as follows:

[0032] The first step is to obtain pig parthenogenetic fetal fibroblasts;

[0033] The pig ovaries are collected from the slaughterhouse, stored in normal saline and transported back to the laboratory, where the pig oocytes are picked and matured in vitro. The mature oocytes are centrifuged at high speed (13000rmp, 20min), so that the fat particles are aggregated into clusters and the fat particles are removed by micromanipulation technology. Then parthenogenetic activation is performed on it to obtain parthenogenetic embryos, and the parthenogenetic embryos are transplanted into surrogate sows that are in estrus on the same day to continue to develop.

[0034] On the 23rd day, the sows were diagnosed with pregnancy, and the 25-day-old surrogate sows were subjected to laparotomy t...

Embodiment 2

[0042] Using somatic cell nuclear transfer technology to continuously clone pig fetal fibroblasts, and finally obtain surviving pig individuals, the specific implementation methods are as follows:

[0043] The first step is to obtain pig parthenogenetic fetal fibroblasts;

[0044] The pig ovaries are collected from the slaughterhouse, stored in normal saline and transported back to the laboratory, where the pig oocytes are picked and matured in vitro. The mature oocytes are centrifuged at high speed (13000rmp, 20min), so that the fat particles are aggregated into clusters and the fat particles are removed by micromanipulation technology. Then parthenogenetic activation is performed on it to obtain parthenogenetic embryos, and the parthenogenetic embryos are transplanted into surrogate sows that are in estrus on the same day to continue to develop.

[0045] On the 23rd day, the sows were diagnosed with pregnancy, and the 25-day-old surrogate sows were subjected to laparotomy t...

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