Improved method used for cloned embryo construction

Abstract

The invention belongs to the field of clone technology, and more specifically discloses an improved method used for cloned embryo construction. The improved method comprises following steps: S1, ovocytes are delivered into an operation droplet A, and blind absorption is adopted so as to realize denucleation; S2, cytoplasm obtained via denucleation is delivered into a dyed droplet, dyeing situations are observed directly under a fluoroscope, and the ovocytes processed via denucleation are reserved for standby application; S3, the ovocytes processed via denucleation are delivered into an operation droplet B for standing, and then are delivered into an operation droplet C containing a donor cells, one of the ovocyte is bound with one donor cell, and then is bound with another ovocyte so as to obtain a somatic cell-ovocyte-ovocyte combination; and S4, the somatic cell-ovocyte-ovocyte combination is allowed to stand in a fusion activation fluid, and then is delivered into a fusion slot for electrofusion. The improved method is simple and rapid in operation; denucleation is complete and high in efficiency; and cloning efficiency is high.

Description

technical field [0001] The invention relates to the technical field of cloning, in particular to an improved method for constructing cloned embryos. Background technique [0002] Animal cloning refers to the process in which animals directly obtain offspring with the same genetic traits as their parents without sexual reproduction, including parthenogenesis, blastomere isolation and culture, embryo segmentation, and nuclear transfer. At present, the well-known animal cloning technology actually refers to the cell nuclear transfer technology. The traditional nuclear transfer technology usually refers to injecting the nucleus of the donor cell into the pre-enucleated mature oocyte or early zygote through micromanipulation to form a new reconstructed embryo. After fusion activation, the reconstructed embryo Developmental reprogramming occurs, through cell division, differentiation and development into a new individual in the surrogate mother. It is a complicated process inclu...

AI Technical Summary

Problems solved by technology

[0005] Blind aspiration and enucleation ( figure 2 ) is based on the characteristics of mature oocytes, removing about 1 / 3 of the cytoplasm near the polar body to achieve the purpose of enucleation. The operation is simple and fast, but the position of the first polar body of the oocyte will change during the enucleation process. Affect the enucleation effect, the enucleation efficiency is relatively low, generally 70%

Staining enucleation uses the DNA-specific dye Hoechest33342 to stain the whole oocyte, and then uses a fluorescent microscope to display the chromosome enucleation method, which can remove a small amount of cytoplasm and ensure 100% cleansing, but ultraviolet radiation will damage embryo viability (Tsunda , 1988)

The spindle-view method developed later uses polarized light refraction to directly obtain the spindle mirror image, and the spindle in the oocyte can be observed without staining. The enucleation rate can reach more than 95%, but the price is expensive, which is not conducive to popularization. application

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