Expression inhibitor of pig SIRT1 gene and application of expression inhibitor

A gene expression and inhibitor technology, applied in the biological field, can solve problems such as low cloning efficiency of pigs, and achieve the effects of improving cloning efficiency, improving developmental performance, and reducing negative effects

Active Publication Date: 2020-04-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the process of studying and solving the problem of low pig cloning efficiency, the inventors of the present invention found that the expression levels of SIRT1 and HDAC2 were significantly positively correlated with developmental arrest at the 2-cell stage of pig cloned reconstituted embryos

Method used

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  • Expression inhibitor of pig SIRT1 gene and application of expression inhibitor
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  • Expression inhibitor of pig SIRT1 gene and application of expression inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Design and synthesis of siRNA of porcine SIRT1 gene and porcine HDAC2 gene

[0042] According to the porcine SIRT1 gene sequence (sequence number: NM_001145750.2) and porcine HDAC2 gene sequence (sequence number: XM_001925318.6), referring to the principle of siRNA design, the corresponding target sites of porcine SIRT1 gene and porcine HDAC2 gene were provided by Shanghai Gemma Pharmaceutical Co., Ltd. Technology Co., Ltd. designed and synthesized 3 pairs of siRNA respectively, which are respectively recorded as SIRT1-siRNA1, SIRT1-siRNA2, SIRT1-siRNA3, HDAC2-siRNA1, HDAC2-siRNA2, HDAC2-siRNA3, and their sequences are shown in Table 1 below:

[0043] Table 1 siRNA sequence

[0044]

[0045] 2. The inhibitory effect of siRNA on genes

[0046] 2.1 siRNA solution preparation

[0047] Centrifuge the tubes containing the dry siRNA powder, and then add 150 μL of sterile, DNase & RNase-free embryo injection-grade water to each tube (purchased from Sigma-Aldrich (Shang...

Embodiment 2

[0070] 1. Effect of EX527 on the developmental performance of pig somatic cell cloned embryos

[0071] The small molecule EX527, which specifically inhibits SIRT1, was added to the culture medium to incubate the reconstituted embryos of 1cell stage pig clones for 24 hours, and then replaced with normal medium to continue the culture. The NC group was supplemented with the same amount of untreated normal medium. The cleavage rate of cloned and reconstituted embryos in the experimental group was observed on day 2 and the blastocyst development rate on day 7, and nuclear fluorescent staining was performed on blastocyst cells, and the total number of cells was counted. The results are shown in Table 3.

[0072] Table 3 Effects of different concentrations of EX527 on the developmental performance of pig somatic cell cloned embryos

[0073]

[0074] From the results in Table 3, it can be seen that compared with the NC group, the cleavage rate of the three concentrations of EX52...

Embodiment 3

[0079] Mix SIRT1-siRNA1, SIRT1-siRNA2, SIRT1-siRNA3, HDAC2-siRNA1, HDAC2-siRNA2, and HDAC2-siRNA3 at a molar ratio of 1:1:1:1:1:1 and inject pig clones into 1 cell for reconstitution Embryos and NC group were injected with the same amount of NC-siRNA solution. The cleavage rate was observed on the second day of culture, and the blastocysts were collected on the seventh day to record the embryo development efficiency, and the blastocyst cells were stained with nuclear fluorescence to observe the number of blastocyst cells. The results are shown in Table 4.

[0080] Table 4 Effects of co-injection of SIRT1-siRNA and HDAC2-siRNA on the developmental performance of pig cloned reconstituted embryos

[0081]

[0082] From the results in Table 4, it can be seen that after the joint injection of SIRT1-siRNA and HDAC2-siRNA, the cleavage rate of pig cloned reconstructed embryos at the 2cell stage was significantly increased (P<0.05), and it was better than the injection inhibition ...

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Abstract

The invention discloses an application of an expression inhibitor of a pig SIRT1 gene in preparation of a product with an effect of improving development performance of a pig clone reconstructed embryo. The expression inhibitor of the pig SIRT1 gene can effectively improve the development rate of the pig cloned and reconstructed embryo and the embryo quality in a blastula period, so that the development performance of the pig cloned and reconstructed embryo can be effectively improved, so that the cloning efficiency of porcine somatic cell cloning can be improved. The expression inhibitor of the pig SIRT1 gene can be selected from at least one of siRNA capable of inhibiting the expression of the pig SIRT1 gene and a compound capable of specifically inhibiting the expression of the pig SIRT1 gene. The invention also provides three siRNAs capable of inhibiting the expression of the SIRT1 gene of the pig.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression inhibitor of porcine SIRT1 gene and its application in preparing a product capable of improving the developmental performance of porcine cloned reconstituted embryos. Background technique [0002] Somatic cell cloning technology, that is, somatic cell nuclear transfer technology, uses somatic cells as nuclear donors to transfer somatic cells into enucleated oocytes by special artificial means (micromanipulation, electrofusion, etc.) to generate reconstructed embryos. After the embryos are activated by in vitro fusion and cultured to a certain stage, they are then transplanted into the surrogate mother so that they can develop into cloned offspring containing the same genetic material as the donor cells. [0003] Pig somatic cell cloning technology has important application value in basic scientific research, animal breeding and medical research, but the over...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6888
CPCC12N15/113C12N2310/141C12Q1/6888C12Q2600/124C12Q2600/158
Inventor 李紫聪吴珍芳王兴旺蔡更元吴宵招华兴杨柳松
Owner SOUTH CHINA AGRI UNIV
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