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.
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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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