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Quantitative cell enucleating method

A nuclear method and cell technology, which is applied in the field of micromanipulation at the cell level, can solve the problems of general enucleation volume range, poor consistency of development results, and low cloning operation efficiency, and achieve the goal of improving consistency and cloning operation success rate Effect

Pending Publication Date: 2018-10-02
NANKAI UNIV
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Problems solved by technology

Blind aspiration enucleation is a commonly used enucleation method at present. It generally needs to absorb one quarter to one third of the amount of cytoplasm near the polar body to achieve enucleation. The range of enucleation volume of this method is very general. The amount of enucleation in the process of nucleation has great randomness, resulting in poor consistency of the later development results of the operation and low efficiency of cloning operations

Method used

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  • Quantitative cell enucleating method
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Examples

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Embodiment 1

[0020] The oocytes used in this example were domestic pig oocytes taken from local slaughterhouses.

[0021] Oocytes are obtained as follows:

[0022] After the ovaries were removed from the slaughterhouse, they were transported to the laboratory within two hours in a thermos filled with normal saline at 35° to 37°. Immediately then wash twice at 37° with sterile saline containing 100 IU / L of penicillin and 50 mg / L of streptomycin. Oocytes were extracted from 2 to 6 mm diameter follicles on the ovary, and the extracted cells were washed three times with TL-Hepes-PVA and incubated for 42 hours in in vitro maturation (IVM) in a 39°, 5% carbon dioxide incubator . After IVM, cells were devitrified with 0.1% hyaluronidase. Finally, the cells were washed three times with M199 to obtain the oocytes used.

[0023] This example uses the NK-MR601 micromanipulator to operate, the device system includes two three-degree-of-freedom manipulator arms, one of which is used to load the ooc...

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Abstract

The invention provides a quantitative cell enucleating method, and belongs to the technical field of cell-level micromanipulation. The quantitative cell enucleating method is characterized in that during blind aspiration enucleation, the cytoplasm of an oocyte is found to tend to have elastic solid properties and the equilibrium pressure during the enucleation is linearly reduced, so that quantitative cell enucleation can be achieved through precise control of the equilibrium pressure during the enucleation process. By the quantitative cell enucleating method, the problem that the enucleationamount is difficult to control during the blind aspiration enucleation is solved.

Description

technical field [0001] The invention belongs to the technical field of micromanipulation at the cell level. Background technique [0002] The enucleation process of cells is a key step in biological operations such as nuclear transfer and cloning. The amount of enucleation directly affects the later developmental outcome of cells. Excessive amount of enucleation will accelerate the abnormal differentiation of recombinant embryos; too little amount of enucleation will cause polyploidy, abnormal cleavage and developmental retardation. Blind suction enucleation, as a common enucleation method at present, generally needs to suck one quarter to one third of the cytoplasm near the polar body to achieve enucleation. The amount of denucleation in the nucleation process is very random, resulting in poor consistency of later developmental results and low cloning efficiency. Therefore, it is necessary to design a quantitative enucleation method for cell enucleation, which is necessa...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609
Inventor 赵新刘曜玮冯泽阳孙明竹崔茂盛
Owner NANKAI UNIV
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