Quantitative denucleation method based on cell orientation

Abstract

The invention relates to a quantitative denucleation method based on a cell orientation. The quantitative denucleation method comprises the following steps of S1, performing finite element modeling ona cell nucleus extraction process to determine the distribution of cytoplasm removal amount required by denucleation under relative positions of cell nucleus-microneedles; S2, staining the cell nucleus, and positioning a three-dimensional distribution range of the cell nucleus relative to a polar body in a fluorescence field; S3, determining a point location of the polar body on a focal plane asthe cell orientation during denucleation operation by combining results of the two steps; and S4, rotating the cells to the direction to suck quantitative cytoplasm for denucleation. According to themethod, the point location of the polar body during denucleation is determined by utilizing finite element modeling in the cell nucleus extraction process and the three-dimensional distribution rangeof the cell nucleus relative to the polar body determined offline, the cytoplasm removal amount is automatically controlled, and the 96% denucleation success rate under the 10% denucleation amount canbe realized.

Description

technical field [0001] The invention relates to the field of cell-level micromanipulation, in particular to a quantitative enucleation method based on cell orientation. Background technique [0002] Cell enucleation refers to the operation of removing the nucleus of the recipient oocyte containing genetic material and is a key step in cloning operations. Excessive loss of cytoplasm during cell enucleation can deprive cloned embryos of excess nutrients, which in turn can lead to embryonic failure or reduced embryonic developmental potential. Therefore, in the enucleation operation, the amount of cell mass removal should be minimized under the premise of complete removal of nuclei. [0003] At present, the existing cell denucleation operations mainly include the following methods: (1) immersing the nucleus with special chemical substances; (2) irradiating the part of the nucleus with laser, X-ray or ultraviolet light to make the nucleus lose its function; (3) using Magnetic-...

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Problems solved by technology

[0003] At present, the existing cell enucleation operations mainly include the following methods: (1) using special chemical substances to immerse the nucleus; (2) irradiating the nucleus with laser, X-ray or ultraviolet rays to make the nucleus lose its function; (3) using Magnetically driven microblades or microfluidic channels are used to cut and enucleate cells in two; when using the above three common methods to enucleate cells, there are mainly the following defects: a. When cells are soaked in special chemicals to achieve functional enucleation, the remaining Chemical substances will be detrimental to the expression of the subsequently introduced donor gene; b. X-ray and laser irradiation of the nucleus area will cause the gene to lose the expression ability of the cell, and the energy generated by the laser and X-ray will destroy the activity of the cytoplasm around the nucleus and reduce the clone Embryo developmental potential; c. Use magnetically driven microblades or microfluidic channels to cut cells in half, and discard the half containing the nucleus can also be enucleated, but the huge loss of cytoplasm makes it difficult to apply to cloning operations

However, this enucleation method mainly has the following problems in the actual application process: due to the darker cytoplasm of livestock oocytes, the nuclei buried in the cytoplasm are generally invisible

However, since the relative position of the nuclei-oblique microneedles and the three-dimensional distribution of the nucleus relative to the polar body required to complete the enucleation with a small amount of enucleation are unknown, it is difficult to determine a specific polar body point for enucleation optimal cell orientation

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