A Quantitative Enucleation Method Based on Cell Orientation

Abstract

The invention relates to a quantitative enucleation method based on cell orientation, comprising the following steps: S1, modeling the finite element method of the cell nucleus extraction process to determine the distribution of cytoplasm removal required for enucleation under the relative position of the nucleus-microneedle; S2 , stain the cell nucleus and locate the three-dimensional distribution range of the cell nucleus relative to the polar body under the fluorescent field; S3, combine the results of the above two steps to determine the point of a polar body on the focal plane as the cell orientation during the enucleation operation; S4, rotate the cell Draw and quantify the cytoplasm in this direction for enucleation. The present invention utilizes the finite element modeling of the cell nucleus extraction process and the off-line determined cell nucleus relative to the three-dimensional distribution range of the polar body to determine the polar body point during enucleation, and automatically controls the amount of cytoplasm removal, which can achieve 96% of the enucleation amount under 10%. enucleation success rate.

Description

technical field [0001] The invention relates to the field of micromanipulation at the cell level, in particular to a quantitative enucleation method based on cell orientation. Background technique [0002] Cell enucleation refers to the operation of removing the nucleus containing genetic material in the recipient oocyte, which is a key step in the cloning operation. Excessive loss of cytoplasm during enucleation can deprive cloned embryos of excessive nutrients, which in turn leads to failure of embryonic development or reduced embryonic developmental potential. Therefore, in the enucleation operation, the removal amount of the cytoplasm should be reduced as much as possible under the premise that the nucleus is completely removed. [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 nucle...

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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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