Seven synthesized shRNA (short hairpin ribonucleic acid) molecules for inhibiting expression of BMPR-1B (bone morphogenetic protein receptor-1B) genes

A gene expression and molecular technology, applied in the fields of seven shRNA molecules that inhibit the expression and synthesis of BMPR-1B gene, can solve the problems of skeletal muscle development defects, multiple birth traits, and cartilage formation effects, and achieve the effect of highlighting technological progress.

Active Publication Date: 2012-05-02
新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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Problems solved by technology

[0003] Regarding the study of BMPR-1B gene shRNA molecules, the mice with ALK6 gene interference did not show multiple fecundity traits like Booroola ewes, and the follicle development and ovulation rate were normal, but their cartil

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Embodiment Construction

[0030]The present invention is further described in conjunction with embodiment.

[0031] According to the sheep BMPR-1B gene in GeneBank, that is, the NM-001009431.1 sequence; design 34 shRNA molecules and synthesize them for shRNA sequences at different sites; Connected to the pLL3.7 lentiviral interference vector, constructed 8 lentiviral interference RNA plasmids, the pLL-BMPR-1B-shRNA interference RNA plasmid and the BMPR-1B expression vector were transfected with liposomes to produce toxins by 293T method. The detection of fluorescent PCR and Western blot determined 7 shRNA molecules that significantly inhibited the expression of BMPR-1B gene; the primers used (omitted);

[0032] 1) Construction of shRNA lentiviral vector

[0033] Take 10UL of 10uM two siRNA oligonucleotides (positive strand and complementary strand) respectively, add 8μL 10×annealing buffer and 12μL sterilized ultrapure water, boil for 10min and cool down to room temperature naturally, at the same time...

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Abstract

The invention provides a synthesized shRNA molecule for inhibiting expression of BMPR-1B (bone morphogenetic protein receptor-1B) genes. According to sheep BMPR-1B genes (a NM_001009431.1 sequence) in a Gene Bank, a shRNA (short hairpin ribonucleic acid) molecule group interfering BMPR-1B gene transcription is designed by shRNA design software, 8 shRNA sequences aiming at different loca are screened from 34 designed shRNA molecules according to structures and positions and are synthesized; two synthesized complementary single-stranded shRNA molecules form double strands after being annealed and are connected to pLL3.7 slow virus interference vectors respectively to construct 8 slow virus interference RNA plasmids (pLL-BMPR-1B-shRNA), the interference RNA plasmids and BMPR-1B expression vectors produce toxin by a liposome transfection 293 T method, and 7 shRNA molecules which obviously inhibit the BMPR-1B genes are obtained after being detected and expressed by a fluorescence PCR (polymerase chain reaction) and a Western blot. The inhibition efficiency reaches more than 90 percent in an mRNA (messenger RNA) level, and the inhibition rate of BMPR-1B proteins reaches more than 80 percent.

Description

technical field [0001] The invention relates to the design, synthesis and evaluation method of the small interference RNA molecule capable of significantly inhibiting the bone morphogenic protein receptor-1B (BMPR-1B) gene and the inhibitory effect on oocytes. Background technique [0002] The BMPR-1B gene is a regulatory gene that increases ovulation rate and belongs to the TGF-? superfamily member, also known as ALK6 gene; A conserved intracellular kinase domain, nucleotide 830 (A830G), causes arginine to replace glutamine (Q249R); its dominant phenotype is early follicular development and multiple births. Literature search disclosure: (1) Biol.Reprod.64, 1225-1235 Author: Wilson, T. et al published the content of the article "A mutation of ALK6 gene expressed in oocytes and granulosa cells in Booroola sheep showing multiple fecundity traits" is to carry Fec B Homozygous ewes had an increased ovulation rate, accompanied by a large number of cystic follicles than Fec B+...

Claims

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Application Information

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IPC IPC(8): C12N15/113
Inventor 黄俊成林嘉鹏金贤华汪立芹白杰刘晨曦
Owner 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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