Liupan mountain yellow cattle circR-UQCC1 gene, and overexpression vector, construction method and application thereof

An overexpression vector, pcd2.1-circr-uqcc1 technology, applied in the field of genetic engineering, can solve problems affecting muscle function, etc.

Active Publication Date: 2019-12-13
GUYUAN BRANCH NINGXIA AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The online software RNAhybrid predicts that circR-UQCC1 can adsorb miR-186, and miR-186 can promote myoblast differentiation by inhibiting myogenin. In addition, miR-186 can inhibit the proliferation of prostate cancer cells, which suggests that circR-UQCC1 is likely to Participate in the proliferation and differentiation of muscle cells, thereby affecting the function of muscles

Method used

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  • Liupan mountain yellow cattle circR-UQCC1 gene, and overexpression vector, construction method and application thereof
  • Liupan mountain yellow cattle circR-UQCC1 gene, and overexpression vector, construction method and application thereof
  • Liupan mountain yellow cattle circR-UQCC1 gene, and overexpression vector, construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Construction of pCD2.1-circR-UQCC1 recombinant overexpression plasmid:

[0034] 1. Materials and methods:

[0035] 1.1. Instrument:

[0036] Ultra-clean bench, biochemical incubator, gene amplification instrument, PTC-200 single-slot gradient gene amplification instrument, Heraeus refrigerated high-speed centrifuge (Germany), Bio-Rad gel imaging analyzer (USA), CO2 incubator, HZS-H water bath shaker (Harbin), Eppendorf pipette, DYY-Ⅲ steady voltage and steady flow electrophoresis instrument (Beijing Liuyi), DYY-Ⅲ31A and DYY-Ⅲ28D electrophoresis tank (Beijing Liuyi), ice maker, MDF-382E ultra-low temperature refrigerator (Sanyo, Japan), Eppendorf desktop high-speed centrifuge, Sartorious electronic balance (Germany), conventional refrigerator, etc.

[0037] 1.2. Main biochemical reagents and kits:

[0038] Long Taq polymerase, PrimeSTAR DNA polymerase, DNA restriction endonuclease (Kpn I and BamH I, etc.), collagen, trypsin, DNA Marker, DNA ligase, Trizol, ...

Embodiment 2

[0077] Example 2: Transfection of the recombinant pCD2.1-circR-UQCC1 plasmid carrying the Liupanshan yellow cattle circR-UQCC1 gene into primary cultured bovine muscle cells:

[0078] The purpose of this embodiment is to: transfect the recombinant pCD2.1-circR-UQCC1 plasmid into bovine primary muscle cells, and detect the effects of overexpression of the circR-UQCC1 gene on muscle cell proliferation and differentiation and muscle development marker genes (MyoD, MyoG) by real-time quantitative PCR. , PCNA, MYHC) differential expression in bovine muscle cells.

[0079] 1. Primary culture of bovine muscle cells

[0080] Place the fetal calf in the operation tray, rinse with sterile PBS containing 1% double antibody for 3 times,

[0081]Cut the epidermal tissue along the back of the fetal bovine, cut out the back muscle and place it in a 6cm culture dish containing 1% double-antibody PBS, use scissors to cut the muscle piece as much as possible, and then collect it into a 50mL ce...

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Abstract

The invention discloses a Liupan mountain yellow cattle circR-UQCC1 gene, and an overexpression vector, a construction method and an application thereof, and belongs to the technical field of gene engineering. The invention discloses the annular Liupan mountain yellow cattle circR-UQCC1 gene for the first time; the recombinant overexpression vector capable of expressing the Liupan mountain yellowcattle circR-UQCC1 gene is constructed; after bovine muscle cells subjected to primary culture are transfected by the constructed vector, efficient expression of circR-UQCC1 mRNA can be obtained, anda foundation is laid for circR-UQCC1 gene function identification, cell transformation, metabolism regulation and growth and development.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a Liupanshan yellow cattle circR-UQCC1 gene and its plasmid-type overexpression vector pcD2.1-circR-UQCC1, a construction method and application thereof. Background technique [0002] At present, epigenetics has become an important research direction for analyzing life activities such as growth and development, cancer development and treatment, and immune response. Regulation of non-coding RNAs is one form of epigenetic regulation. Circular RNA is a class of covalently closed circular non-coding RNA molecules without a 5' end cap and a 3' end poly tail structure. In recent years, with the rapid development of bioinformatics and the continuous innovation of high-throughput sequencing technology, a large number of circRNAs with stable endogenous structure, high abundance and specific spatiotemporal expression have been discovered in eukaryotic cells, indicating that cir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12Q1/6888
CPCC12N9/0004C12N15/70C12Q1/6888C12Q2600/124C12Y110/02002
Inventor 蔡翠翠王健李玉莲万鹏谢建亮杨雪瑶李毓华余治家贾宝光佘萍王维
Owner GUYUAN BRANCH NINGXIA AGRI & FORESTRY SCI
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