Method for enucleating mammal oocyte

An oocyte and mammal technology, applied in the field of embryo engineering, can solve the problems of prolonged operation time, inability to change too much suction, unfavorable large-scale operation, etc., to avoid direct irradiation damage and improve efficiency.

Active Publication Date: 2019-02-12
WENS FOODSTUFF GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the efficiency of enucleation is detected by fluorescent staining after blind aspiration, and it is impossible to change the excessive absorption of cytoplasm by blind aspiration method. Excessive loss of cytoplasm will affect the developmental potential of embryos. The mother cells have to be moved to other droplets for fluorescent staining detection, and if the enucleation is not clean, the enucleation needs to be repositioned, resulting in prolonged operation time, which is not conducive to large-scale operation

Method used

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  • Method for enucleating mammal oocyte
  • Method for enucleating mammal oocyte
  • Method for enucleating mammal oocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 The enucleation method of oocyte of the present invention

[0048] Taking pig oocytes as an example, the following steps are included:

[0049] (1) Obtain the cumulus oocyte complex; at 38.5°C, 5% CO 2 1. In vitro maturation culture for 44 hours under the condition of saturated humidity.

[0050] (2) Transfer the mature oocytes in (1) to a centrifuge tube containing 0.5mL hyaluronidase, blow gently with a pipette gun, and remove the cumulus granulosa cells around the zona pellucida of the oocytes, leaving oocyte;

[0051] (3) Incubate the oocytes obtained in (2) in 0.5 μg / mL decarboxycolchicine (DEME) culture solution for 1 hour to develop nuclei, or treat them in a 5% sucrose solution for 10 minutes to make the oocyte surface form tiny protrusions;

[0052] (4) Place the oocytes after nucleation in (3) in a culture solution containing 7.5 μg / mL Hochest33342 for 2 minutes;

[0053] (5) Adjust the fluorescent lamp: adjust the irradiation range of the fl...

Embodiment 2

[0063] Referring to the enucleation method of Example 1, during the step 7 enucleation operation, the routine group adopts the conventional blind aspiration method (that is, the routine group adopts blind aspiration enucleation according to steps 1, 2, 6, 7, and 7 of Example 1), and improves The group adopted the enucleation method of the present invention, and transplanted the constructed embryos into recipient sows respectively. The results are shown in Table 1, which shows that nuclear transfer using the improved method increases the farrowing rate of recipient sows from 20.83% to 50%. And the live piglets per litter increased from 2.4 to 3. It was shown that this method effectively improves the final efficiency of in vivo development of cloned embryos.

[0064] Table 1 Conventional blind aspiration method and the statistics of the embryo transfer situation that the method of the present invention constructs

[0065] group

[0066] In the oocyte enucleation metho...

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Abstract

The invention belongs to the technical field of embryo engineering, and particularly relates to a method for enucleating mammal oocyte. The method comprises the following steps: obtaining cumulus oocyte compositelex; removing cumulus granulose cells at the periphery of an oocyte transparent zone in the cumulus oocyte compositelex, and leaving oocyte; incubating the obtained oocyte to nucleate; putting the nucleated oocyte into culture solution liquor to treat; debugging a fluorescent lamp; cleaning the oocyte treated by the culture solutionliquor, and transferring the cleaned oocyte into enucleatingstoning liquid drops; and enucleating under fluorescence. The method is simple and convenient to operate, greatly reduces loss of cytoplasm, ensures that each constructed clone embryo loses a little cytoplasm and achieves 100% enucleating, and improves developmental potentiality of the embryos.

Description

technical field [0001] The invention belongs to the technical field of embryo engineering, and in particular relates to a method for enucleation of mammalian oocytes. Background technique [0002] Mammalian somatic cell nuclear transfer, that is, somatic cell cloning, is an epoch-making breakthrough in the field of modern biotechnology and life sciences. and other fields have broad application prospects. In animal nuclear transfer operations, the first step is to remove the nuclear material of the oocyte. Whether the enucleation is complete is one of the key links for the success of nuclear transfer. Incomplete enucleation will lead to the fusion of oocytes and donor nuclei. The number of chromosome sets in the reconstructed embryo is abnormal, and eventually the embryo will be aborted or die due to developmental obstruction. [0003] At present, the enucleation method commonly used in mammalian somatic cell nuclear transfer is physical mechanical enucleation, that is, usi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075C12N15/873
CPCC12N5/0609C12N15/873
Inventor 麦然标石俊松周荣余婉娴罗绿花蔡更元吴珍芳
Owner WENS FOODSTUFF GRP CO LTD
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