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RNA interference method for suppression of gene expression

A technology of RNA interference and gene expression, applied in the field of genetic engineering, can solve problems such as slow start-up speed, and achieve rapid and long-lasting effects

Active Publication Date: 2018-03-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] It usually takes about four days for mouse in vitro fertilized eggs to develop into blastocysts. For gene knockdown in early embryos of mice, people often choose siRNA, but for early embryos of large animals, the time for in vitro culture is relatively short. It takes a long time (about seven or eight days), and ordinary siRNA often cannot degrade target genes continuously and efficiently. Although shRNA has a long-lasting effect, it takes a certain amount of time to transcribe, and the start-up speed is slow

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  • RNA interference method for suppression of gene expression
  • RNA interference method for suppression of gene expression
  • RNA interference method for suppression of gene expression

Examples

Experimental program
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Embodiment 1

[0035] Example 1 Expression difference of NLRP7 gene in sheep tissues

[0036] 1. Extraction of total RNA from different tissues

[0037] 2. Reverse transcription of RNA RNA removal genome, kit PrimeScript TM RT reagent Kit with gDNAEraser (Perfect Real Time) was purchased from Takara Company.

[0038] 3. Design and synthesis of primers In the present invention, the published or predicted NLRP7 gene coding region sequences of humans, goats, cattle and other animals are compared with the predicted sequence of sheep NLRP7 by NCBI and compared with DNAMAN software to find out their shared conserved parts , and then use Primer Premier 5.0 software to design primers based on the consensus region of the predicted sequence of sheep NLRP7. The primers were synthesized by Beijing Sangon Biotechnology Co., Ltd., and the primer sequences are as follows:

[0039] Sense strand: 5'-TGCTTACCGGGACTTCTGTC-3' (SEQ ID No: 1 in the sequence listing);

[0040] Antisense strand: 5'-CCACTGCCAAGT...

Embodiment 2

[0048] Example 2 Expression, localization and cloning of NLRP7 gene in sheep ovary

[0049] This example aims to ascertain the expression and localization of NLRP7 protein in sheep ovary tissue by immunohistochemistry, and at the same time, sheep NLRP7 is cloned by RT-PCR using sheep cumulus oocyte body as material. 1. Preparation of Paraffin Sections

[0050] 2. Immunohistochemical staining and histochemical kit were purchased from Zhongshan Jinqiao Company, Cat. No. PV-9001. Immunohistochemical results such as Figure 5 As shown, the figure shows primordial follicles (Panel A), primary follicles (Panel B), secondary follicles (Panels C, D), tertiary follicles (Panels E, F) and mature follicles (Panel G) located in the ovarian cortex Figure 1), the negative control (Figure H), the staining situation, it can be seen that the cytoplasm of oocytes at all levels is deeply stained with brown-yellow granules, and the follicle cells surrounding the oocytes are also positively stai...

Embodiment 3

[0058] Example 3 Effect of knockdown of NLRP7 gene on early embryonic development of sheep

[0059] RNA interference usually uses siRNA, which is widely used because it can act quickly in a short period of time. However, because siRNA is easily degraded, it does not last for a long time. However, the early embryonic development of sheep usually lasts for about a week. Ordinary siRNA is very slow. It is difficult to target and degrade mRNA all the time, and its efficacy is greatly reduced at the later stage of embryonic development, often failing to achieve the effect of continuous knockdown of the target gene. shRNA can be continuously synthesized by host cells and spliced ​​into siRNA, so it has a long-lasting effect The shortcoming is that the shRNA interference plasmid does not work immediately after entering the host cell, but takes a certain amount of time to transcribe and translate, and uses the endogenous Dicer enzyme to produce siRNA and then targets and degrades the t...

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Abstract

The invention provides an RNA interference method for suppression of gene expression, wherein target gene siRNAs and a shRNA-containing plasmid are simultaneously microinjected into a host cell wherethe target gene is located. The invention further provides the improved RNA interference method for suppression of sheep early parthenogenetic embryo NLRP7 gene expression, an NLRP7 gene siRNA interference fragment and an shRNA interference plasmid are mixed and injected into a mature oocyte, the siRNAs are shown as SEQ ID NO.6-7, 8-9 and / or 10-11, and the shRNA is shown as SEQ ID NO.14, 16 and / or18; after parthenogenetic activation, an embryo after the NLRP7 gene is knocked down is found to have abnormal development, and the NLRP7 is indicated to be a key gene required for normal developmentof the sheep early parthenogenetic embryo. The method provides a new method for research of gene functions.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an RNA interference method for inhibiting gene expression, in particular to an RNA interference method for specifically inhibiting NLRP7 gene expression. Background technique [0002] NLRs (Nucleotide-binding domain and leucine-rich repeat containing) is the NOD-like receptor family, which is one of the many complex immune recognition systems of organisms. It is divided into many subfamilies according to their structural domains. Among them, pyrin will be included The subfamilies of domains are called NLRP subfamilies. The NLRP gene family was first discovered in humans, with a total of fourteen members named NLRP1-NLRP14. There are also fourteen members in primates, which are named the same as humans. Compared with the human NLRP gene family, there are no NLRP7, NLRP8, NLRP11, and NLRP13 in the mouse NLRP gene family, but NLRP1, NLRP4, and NLRP9 have changed duri...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/85C12N15/113
CPCC07K14/705C12N15/1138C12N15/85C12N15/89C12N2310/14C12N2800/107C12N2310/531
Inventor 刘国世李广栋崔炜田秀芝姬鹏云吕东颖吴昊柴孟龙连正兴阿布力孜·吾斯曼
Owner CHINA AGRI UNIV
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