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Method for cloning animal somatic cell

A technology of somatic cell cloning and cloning method, which is applied in the field of animal somatic cell cloning, can solve problems such as polyhydramnios or giant fetus, fetal development delay, fetal death, etc., reduce miscarriage rate and dystocia rate, increase pregnancy rate, improve quality effect

Active Publication Date: 2008-05-21
保定国农温氏种猪育种股份有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] Through the follow-up observation of cloned embryos transplanted into recipients, it is found that most of the embryos can implant and develop after transplantation, and the pregnancy rate is not much different from that of in vitro fertilization (IVF) embryos, but there is a difference after 2-3 months. 40% of the fetuses are aborted, and about 1 / 3 of the unaborted fetuses have obviously delayed development, which eventually leads to fetal death. Among the embryos that can continue to develop, there are still quite a few recipients with polyhydramnios or macrofetal phenomenon , the above situation shows that there is a big problem in the material transportation between the fetus and the mother of the cloned animal. In the process of preparing the cloned cattle, our laboratory has also found many times that the fetus of the cloned cattle was stillborn because of the placenta ulceration. It can be seen that the placenta Developmental abnormalities are important cause of developmental failure in cloned animals

Method used

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  • Method for cloning animal somatic cell
  • Method for cloning animal somatic cell

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Example 1. Cloned cattle

[0017] 1. Donor - preparation of tetraploid blastomeres (4n)

[0018] 1.1 In vitro maturation of oocytes

[0019] The slaughterhouse collects the ovaries of adult cattle and puts them in 30°C normal saline and sends them to the laboratory within 4 hours. Wash the ovaries three times in 37°C PBS solution and put them in a container. Select follicles with a diameter of 4-8mm Use a 18-gauge needle of a 10ml syringe to draw follicular fluid, classify the recovered oocytes, select a type A cumulus oocyte complex with more than three layers of dense cumulus, and wash 3 times with maturation solution (maturation solution is M199+ 10% FBS+0.01U / mLbFSH+0.01U / mLbLH+1μg / mL estradiol), then put 30-40 pieces / drop of cumulus oocyte complex into the mature liquid drop, at 38.5℃ in 5% CO 2 Cultivate and mature in the incubator for 22hrs.

[0020] 1.2 Thawing of frozen semen and capacitation in vitro

[0021] Thaw the frozen semen of Holstein cattle from t...

Embodiment 2

[0048] Example 2. Blastomeres from tetraploid in vitro fertilization (IVF) are put into cloned embryos at the 8-16 cell stage to develop into trophoblast cells

[0049] To illustrate this process, the cloned blastomeres with enhanced green fluorescent protein (EGFP) were used as the marker signal to replace the ordinary IVF blastomeres. The specific operation is as follows, as shown in Figure 2:

[0050] The plasmid pEGEP-N1 (GibcoBRL company, catalog number: 6085-1) containing the EGFP gene was transferred into the fibroblast cell line prepared from Holstein cows according to step 2.1 of Example 1, and used as the donor cell line according to Example 1. Steps 2.1-2.5 were activated and divided to the 2-cell stage, and then obtained tetraploid blastomeres (4n) according to step 1.4 of Example 1, and cultivated to the 8-cells stage. The 8-cells stage tetraploid blastomeres (4n) transfected with EGFP gene were transferred into the 8-cell stage cloned embryos prepared according t...

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Abstract

The invention discloses a cloning method of the animal somatic cells. The cloning method of the animal somatic cells is that tetraploid blastomere in 8-16 cell stage which is obtained from vitro fertilization is transferred into a somatic cloned embryo in 8-16 cell stage to get a compound embryo; the compound embryo is cultured to get a cloned animal. The invention initiatively changes the sourceof trophoblast cell; the tetraploid blastomere obtained from vitro fertilization is put into the somatic cloned embryo in 8-16 cell stage, which can be developed into trophoblast cell; then the placenta quality is improved, the development condition for the fetus is promoted, the pregnancy rate of the cloned embryo is increased, the abortion rate and the dystocia rate is decreased and the cloningefficiency of the somatic cell is greatly improved. As the fetus is constituted by ploid cells, the tetraploid blastomere can only develop into the placenta without the possibility of developing intofetus. Therefore, the method does not affect the individual cloned animal.

Description

technical field [0001] The invention relates to a method for cloning animal somatic cells. Background technique [0002] Animal somatic cell cloning is also called somatic cell nuclear transfer, which is to transfer the somatic cells of animals into mature oocytes with chromatin removed by the method of nuclear transfer after synchronizing treatment, and then develop into cloned embryos in vitro after activation And transplanted into recipient animals, the method of producing cloned animals after pregnancy. Theoretically speaking, this method can clone individual animals without limitation, because the various somatic cell nuclei of differentiation are inexhaustible. The birth of the cloned sheep "Dolly" is a milestone in the history of cloning technology. It theoretically proves that highly differentiated animal cells still have the totipotency to develop into a complete organism, and may develop into a new individual through genome reprogramming under appropriate conditi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N15/873
Inventor 戴蕴平李宁刘颖王莉莉王海平李荣丁方荣李京李松
Owner 保定国农温氏种猪育种股份有限公司
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