Method for obtaining male pronucleus by using xenogeneic animal oocytes as medium

A technology of oocytes and male pronuclei, which is applied in the field of embryo engineering, can solve the problems that hinder the development of cloning technology, oversize, immune disorders, etc., and achieve the effect of improving the rate of in vitro fertilization, increasing the utilization rate, and high utilization rate

Active Publication Date: 2020-01-14
武汉纽利特生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the low success rate of cloning technology at the present stage hinders the development of cloning technology to a great extent. In addition, many clon...

Method used

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  • Method for obtaining male pronucleus by using xenogeneic animal oocytes as medium

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Obtain mature oocytes from Kunming mice; the Kunming mice used in this example were purchased from the Wuhan Center for Disease Control and Prevention, and 6-10 week-old Kunming white female mice were fed with light control (14h light, 10h darkness) for 1-2 weeks. Intraperitoneal injection of 10IU / branch of PMSG for 46-48h, intraperitoneal injection of 10IU / branch of hCG for superovulation. After 20-22 hours of superovulation, the superovulated mice were sacrificed by cervical dislocation, and the enlarged part of the ampulla was dissected to obtain superovulated oocytes, which were observed under an inverted microscope to obtain mature oocytes with the first polar body.

[0030] Acquisition of high-quality sperm from high-yielding dairy cows: The semen used in this example is frozen Simontal cow semen, which was purchased from Wuhan Dairy Cow Improvement Station. Use upward floating hair to obtain high-quality sperm. The specific operation method: take a thin tube of f...

Embodiment 2

[0034] The difference between this example and Example 1 is that the mature oocytes of Kunming mice were placed in the mouse in vitro fertilization medium HTF to balance for 30 minutes, and the high-yielding dairy cow semen was treated with the mouse in vitro fertilization fluid HTF, and the in vitro fertilization microdrops were used HTF, in vitro fertilization conditions Use mouse in vitro fertilization conditions for egg incubation: 37°C, 5% CO 2 , saturated humidity.

Embodiment 3

[0036] The Kunming mouse oocytes obtained in Example 1 with high-yielding dairy cow male pronuclei are washed three times with cow embryo culture medium CR1aa and then placed in cow embryo culture medium CR1aa, and cultivated using the cow embryo culture conditions: 39°C, 5% CO 2 , saturated humidity.

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Abstract

The invention relates to the technical field of embryo engineering, in particular to a method for obtaining male pronucleus by using xenogeneic animal oocytes as a medium. The method comprises the following steps of step 1, obtaining mature oocytes of animals A, with a first polar body, and placing the obtained mature oocytes in external fertilization liquid droplets of animals B; step 2, injecting the sperms of the animals B into the liquid droplets containing the oocytes in the step 1 to obtain sperm and oveum mixed droplets; and step 3, placing the sperm and oveum mixed droplets obtained inthe step (2) in a CO2 incubator, and performing hatching until sperms of the animals B enter the cytoplasm of the oocytes to form male pronucleus, wherein the animals A and the animals B are xenogeneic animals. According to the method, a fertilization biology principle of reproductive isolation of uncrossed fertilization of the xenogeneic animals is used, an external fertilization method is adopted, the oocytes of the xenogeneic animals are used as a medium to obtain the male pronucleus of another animal, and the formation rate of the male pronucleus is as high as 17.8% or above.

Description

technical field [0001] The invention relates to the technical field of embryo engineering, in particular to a method for obtaining male pronuclei by using oocytes among heterogeneous animals as a medium. Background technique: [0002] In vitro fertilization refers to the technology in which mammalian sperm and eggs complete the fertilization process in an artificially controlled environment outside the body. This technology was successfully developed in the 1950s and has developed rapidly in the past 20 years. Now it has become an important and routine animal reproduction biotechnology. Biotechnology, such as cloning, transgenic, embryonic stem cell separation and culture and sex control, etc., can provide sufficient embryo sources for the introduction of foreign genes through in vitro fertilization, and provide mature oocytes and cloned embryo culture for cloning techniques System, using separated X and Y sperm to fertilize eggs in vitro, can control the gender of mammals....

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2517/00
Inventor 张立苹林郑云郑新民毕延震华再东肖红卫任红艳华文君
Owner 武汉纽利特生物技术有限公司
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