Method for improving development efficiency of pig cloned embryos

A technology for cloning embryos and high efficiency, which is applied in the field of developmental efficiency of pig cloned embryos, and can solve problems such as the impact on the developmental potential of pig cloned embryos, which has not been reported.

Active Publication Date: 2020-04-28
WENS FOOD GRP CO LTD
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  • Claims
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AI Technical Summary

Problems solved by technology

At present, more than ten kinds of deubiquitinating enzymes at specific sites of histone H2A and H2B have been discovered, and studies have shown that some types of deubiquitinating enzymes are also involved in the regulation of histone lysine methylation and DNA methylation Therefore, it may also play an important role in regulating the early development of cloned embryos (Pinto-Fernandez and Kessler, 2016), but there is no related report on the influence of histone ubiquitination on the developmental potential of pig cloned embryos.

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  • Method for improving development efficiency of pig cloned embryos
  • Method for improving development efficiency of pig cloned embryos
  • Method for improving development efficiency of pig cloned embryos

Examples

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Embodiment 1

[0032] Example 1 Combined regulation of histone ubiquitination, H3K9me3 and H3K4me3 modification levels during early development of pig cloned embryos

[0033] 1. Acquisition of porcine in vitro matured oocytes

[0034] Pig ovaries were collected from the slaughterhouse, put into a thermos bottle filled with normal saline, and transported to the laboratory. The follicular fluid was aspirated with a sterile syringe, and then the cumulus cells were selected under a stereoscopic microscope to tightly wrap more than three layers, and the cytoplasm Uniform oocytes. in CO 2 After culturing in the incubator for 42-44 hours, suck out the oocytes and put them into a 1.5ml centrifuge tube containing 200μl of hyaluronidase, and then repeatedly blow and beat to remove the surrounding granulosa cells. The oocytes with obvious polar bodies were selected under a stereomicroscope to be used as nuclear transfer recipients.

[0035] 2. Isolation and culture of porcine cloned donor cells

[00...

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Abstract

The invention belongs to the field of animal somatic cell cloning methods, and in particular relates to a method for improving development efficiency of pig cloned embryos. A main idea of the method provided by the invention is to jointly regulate histone ubiquitination of the cloned early embryos, and modification degrees of histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 4 trimethylation (H3K4me3) to improve the development efficiency of the pig cloned embryos, wherein the combined regulation refers to simultaneous reduction of the histone ubiquitination and the modification degrees of the H3K9me3 and the H3K4me3. The method provided by the invention comprises the following main operation steps: obtaining the pig somatic cell cloned embryos; obtaining exogenous mRNA; and introducing the exogenous mRNA into the cloned embryos. The method provided by the invention simultaneously overexpresses USP29, KDM4A and KDM5B genes, significantly improves the blastocyst rate ofdevelopment of the pig cloned embryos and the total number of cells in blastocyst, and improves the birth efficiency of cloned pigs by 3.82 times.

Description

technical field [0001] The invention belongs to the field of animal somatic cell cloning methods, and specifically relates to a method based on joint regulation of histone ubiquitination, trimethylation (H3K9me3) on the 9th lysine of histone H3 and 4th lysine of histone H3 A method to increase the degree of trimethylation (H3K4me3) modification to improve the developmental efficiency of porcine cloned embryos. Background technique [0002] Somatic cell cloning, also known as somatic cell nuclear transfer, refers to injecting the nucleus of an animal somatic cell into an enucleated oocyte to form a recombined cloned embryo and restore it to cell division. The process of transplanting into the uterus of a surrogate mother to produce offspring with the same genotype as the donor cell after completing the full development period. This technology has great application value in agricultural and medical applications such as replicating superior breeding stock, producing transgenic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N15/53C12N15/87A01K67/027
CPCC12N9/485C12N9/0071C12N15/8778A01K67/0275A01K2227/108
Inventor 吴珍芳贺晓燕石俊松谈成罗绿花余婉娴
Owner WENS FOOD GRP CO LTD
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