New method for gene injection for somatic cell nuclear transfer reconstructed embryo

A technology of somatic cell nuclear transfer and a new method, applied in the field of somatic cell nuclear transfer recombinant embryo injection gene, can solve the problems of non-compliance with the safety requirements of genetically modified organisms, difficulty in screening, etc.

Inactive Publication Date: 2017-03-22
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of SCNT technology to produce transgenic pigs faces two major bottlenecks. One is that no porcine embryonic stem cell lines with germline chimerism have been isolated so fa

Method used

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Examples

Experimental program
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Embodiment 1

[0027] A new gene injection method for somatic cell nuclear transfer recombinant embryos, comprising the following steps:

[0028] Step 1, preparation of target gene

[0029] Obtain the target gene and construct the expression vector;

[0030] Step 2. Isolation and culture of donor cells

[0031] Take out the fetus from the uterus, wash the fetus with DPBS containing antibiotics, transfer to the ultra-clean workbench, remove the fetal head, limbs, internal organs with scissors, rinse with DPBS; cut the remaining part with scissors in a cell culture dish with a diameter of 100mm, and tissue pieces size is 1mm 3 , add a little serum, transfer the tissue block to the bottom wall of the cell culture bottle; spread the tissue block evenly with an elbow pipette, put the side covered with the tissue block facing up, add 15mL of cell culture medium, and then put it in 39℃ , relative humidity is 100%, volume fraction is 5% CO 2 Incubator; after culturing for 6 hours, turn over the ...

Embodiment 2

[0040] A new gene injection method for somatic cell nuclear transfer recombinant embryos, comprising the following steps:

[0041] Step 1, preparation of target gene

[0042] Obtain the target gene and construct the expression vector;

[0043] Step 2. Isolation and culture of donor cells

[0044] Take out the fetus from the uterus, wash the fetus with DPBS containing antibiotics, transfer to the ultra-clean workbench, remove the fetal head, limbs, internal organs with scissors, rinse with DPBS; cut the remaining part with scissors in a cell culture dish with a diameter of 100mm, and tissue pieces size is 1mm 3 , add a little serum, transfer the tissue block to the bottom wall of the cell culture bottle; spread the tissue block evenly with an elbow pipette, put the side covered with the tissue block facing up, add 15mL of cell culture medium, and then put it in 39℃ , relative humidity is 100%, volume fraction is 5% CO 2 Incubator; after 7 hours of culture, turn over the sid...

Embodiment 3

[0053] A new gene injection method for somatic cell nuclear transfer recombinant embryos, comprising the following steps:

[0054] Step 1, preparation of target gene

[0055] Obtain the target gene and construct the expression vector;

[0056] Step 2. Isolation and culture of donor cells

[0057] Take out the fetus from the uterus, wash the fetus with DPBS containing antibiotics, transfer to the ultra-clean workbench, remove the fetal head, limbs, internal organs with scissors, rinse with DPBS; cut the remaining part with scissors in a cell culture dish with a diameter of 100mm, and tissue pieces size is 1mm 3 , add a little serum, transfer the tissue block to the bottom wall of the cell culture bottle; spread the tissue block evenly with an elbow pipette, put the side covered with the tissue block facing up, add 15mL of cell culture medium, and then put it in 39℃ , relative humidity is 100%, volume fraction is 5% CO 2 Incubator; after culturing for 8 hours, turn over the ...

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Abstract

The invention discloses a new method for gene injection for a somatic cell nuclear transfer reconstructed embryo. The method comprises the following steps: target gene preparation, somatic cell nuclear transfer and a microinjection technology. An expression vector is constructed after a target gene is obtained, the cell nucleus of an oocyte is removed by virtue of micromanipulation, then a somatic cell is injected in the space of the denucleated oocyte, then an exogenous gene is directly injected in the somatic cell, the reconstructed embryo is constructed through one-step electric fusion and activation, and the embryo is transferred to obtain a transgenic progeny. According to the method disclosed by the invention, large-fragment cell gene transfection and screening are avoided, the transgenosis efficiency is increased, the time is saved, and the transgenosis application range is expanded. Importantly, the somatic cell is injected in a transpanent zone during a nuclear transfer operation process, thus microinjection for the exogenous target gene is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new gene injection method for recombinant embryos of somatic cell nuclear transfer. Background technique [0002] Somatic cell nuclear transfer (SCNT), also known as somatic cell cloning, is to transfer the donor cell nucleus into the enucleated oocyte to reconstitute a new embryo, which is activated without sperm penetration and other sexual processes , Split and develop, so that the gene of the nuclear donor is completely replicated, and after in vitro culture, the well-developed embryo is transplanted into the animal body. The final product obtained by somatic cell nuclear transfer is called a cloned animal. Mammalian cell nuclear transfer research has not been a long time, marked by the birth of "Dolly the sheep" in the Wilmut laboratory of the Roslin Institute in the United Kingdom in 1997, which opened the prelude to somatic cell nuclear transfer. Somatic cell n...

Claims

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Application Information

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IPC IPC(8): C12N15/877C12N15/89A01K67/027
CPCC12N15/8778A01K67/0278A01K2227/108C12N15/89
Inventor 华再东郑新民任红艳李莉毕延震张立苹肖红卫刘西梅
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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