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A method for enucleating mammalian oocytes

A technology for oocytes and mammals, applied in the field of embryo engineering, can solve the problems of prolonged operation time, unable to change too much absorption, unfavorable large-scale operation, etc., and achieve the effect of reducing the irradiation range

Active Publication Date: 2022-05-27
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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  • A method for enucleating mammalian oocytes
  • A method for enucleating mammalian oocytes
  • A method for enucleating mammalian oocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Enucleation method of oocyte of the present invention

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

[0049] (1) Obtain the cumulus-oocyte complex; at 38.5°C, 5% CO 2 , Under the condition of saturated humidity, it was matured and cultured in vitro for 44h.

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

[0051] (3) Incubate the oocytes obtained in (2) in 0.5 μg / mL decarboxycolchicine (DEME) medium for 1 h, or in 5% sucrose solution for 10 min, so that the oocytes surface formation of tiny protrusions;

[0052] (4) The oocytes after nucleation in (3) were placed in a culture medium containing 7.5 μg / mL of Hochest33342 for 2 min;

[0053] (5) Debugging the fluorescent lamp: Adjust the illumination range of the fluorescent lamp to t...

Embodiment 2

[0063] Referring to the denucleation method of Example 1, during the denucleation operation in step 7, the conventional group adopts the conventional blind suction method (that is, the conventional group adopts the blind suction and denucleation according to steps 1, 2, 6, 7, and 7 of Example 1), and the improved The group used the enucleation method of the present invention to transfer the constructed embryos into the recipient sows respectively. The results are shown in Table 1. And the number of live cubs per litter increased from 2.4 to 3. It is shown that this method effectively improves the final efficiency of in vivo development of cloned embryos.

[0064] Table 1 Statistics of embryo transfer situation constructed by conventional blind suction method and the method of the present invention

[0065] group total number of embryos Number of recipient pigs Number of pigs farrowing total litter size total live births birth rate live cubs regula...

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Abstract

The invention belongs to the technical field of embryo engineering, and in particular relates to a method for enucleation of mammalian oocytes. The method of the present invention comprises the following steps: obtaining a cumulus oocyte complex; removing the cumulus granulosa cells on the periphery of the oocyte zona pellucida in the cumulus oocyte complex, leaving oocytes; incubating the obtained oocytes Nucleation; put the nucleated oocytes in the culture medium for treatment; adjust the fluorescent lamp; wash the oocytes treated with the culture medium and transfer them to the enucleation droplet; The method of the invention is easy to operate, greatly reduces the loss of cytoplasm, ensures that each constructed cloned embryo loses little cytoplasm and is 100% completely enucleated, and improves the developmental potential of the embryo.

Description

technical field [0001] The invention belongs to the technical field of embryo engineering, in particular to a method for enucleating 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 from the oocyte. Whether or not enucleation is complete is one of the key steps in the success of nuclear transfer. The number of chromosomes in the reconstructed embryos is abnormal, and the embryos eventually abort or die due to developmental arrest. [0003] At present, the enucleation method commonly used in mammalian somatic cell nuclear transfer is physical mechanical enucleation, that is, a specially processed capillary glass tube is inserted into the MII oocyte by micromanipulation, and the nu...

Claims

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Application Information

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Patent Type & Authority Patents(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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