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Bovine CFL2 gene adenovirus interference vector and construction and identification method thereof

A technique for interfering with vectors and construction methods, which is applied in the field of genetic engineering and can solve problems such as knocking down CFL2 gene expression

Pending Publication Date: 2022-04-08
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a bovine CFL2 gene adenovirus interference vector and its construction and identification method, aiming to solve the problem of sustained knockdown of CFL2 gene expression in bovine muscle tissue and skeletal muscle cells

Method used

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  • Bovine CFL2 gene adenovirus interference vector and construction and identification method thereof
  • Bovine CFL2 gene adenovirus interference vector and construction and identification method thereof
  • Bovine CFL2 gene adenovirus interference vector and construction and identification method thereof

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Experimental program
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Effect test

Embodiment 1

[0060] The sequence design of the bovine CFL2 gene adenovirus interference carrier provided by the implementation of the present invention is as follows:

[0061] According to the sequence (NC_037348.1) of the CFL2 gene published by GenBank and the pENTR / CMV-GFP / U6 plasmid map ( figure 1 ), a BamH I (GGATCC) restriction site is added to the 5' end of the upstream primer, an Xho I (CTCGAG) restriction site is added to the 5' end of the downstream primer, and a Loop sequence (gAGTACTg, Loop reverse complementary on both sides of the sequence), add a tttttt termination signal at the end, and add sticky ends for ligation (G on the BamH I restriction site, C on the Xho I restriction site), and design negative control primers. The specific primer sequences are as follows:

[0062] The sequence of bovine CFL2 shRNA-1 is SEQ ID NO: 1 (5'-3'):

[0063] shRNA-1 upstream primer: gatccCTGAAAGTGCACCGTTAAAgAGTACTgTTTAACGGTGCACTTTCAG t ttttt C (SEQ ID NO: 1)

[0064] shRNA-1 downstrea...

Embodiment 2

[0128] Example 2 Production Experimental Process of Recombinant Adenovirus Ad-CFL2 shRNA Adenovirus

[0129] 1. Packaging of Recombinant Adenoviral Vectors

[0130] When the growth density of HEK293A cells reached 70%, the recombinant adenovirus vector plasmid pENTR / CMV-GFP / U6-shRNA and backbone plasmid pAd / PL-DEST were taken and transfected with Lipo 2000 transfection reagent. The specific steps are:

[0131] a. Replace the complete medium 2 hours before transfection. Take 2ug recombinant adenovirus vector plasmid pENTR / CMV-GFP / U6-shRNA, and 4ug backbone plasmid pAd / PL-DEST. Dilute with 300 μl of DMEM culture solution and place at room temperature for 5 minutes.

[0132] b. Take 15ul of Lipo 2000, dilute it with 300ul of DMEM culture solution, and place it at room temperature for 5min.

[0133] c. Mix the two, and place in the dark for 20 minutes at room temperature. The mixture was then added to a 60mm Petri dish, shaken in the figure of 8 and placed at 37°C with 5% CO ...

Embodiment 3

[0164] Example 3 Harvesting and Propagation of Recombinant Adenovirus Ad-CFL2 shRNA

[0165] 1. Harvest of recombinant adenovirus vector (P1):

[0166] When the bovine primary muscle cells grew to about 80%, the infection interference effects of Ad-CFL2 shRNA-1 and Ad-CFL2 shRNA-2 were compared, and the results were as follows Figure 5 As shown, the high-titer virus Ad-CFL2shRNA-1 has a better infection interference efficiency. After transfection, the cells were observed daily for signs of cytotoxicity. The phenomenon of poisoning was that the cells became larger and rounder, in the shape of grapes, and obvious plaques began to appear. Wait until most of the cells are diseased and come off from the bottom to collect the poison. Collect all the cells and culture medium in the 60mm culture dish into a 15ml centrifuge tube.

[0167] Turn on the constant temperature water bath to 37°C, freeze and thaw the 15ml centrifuge tube three times in liquid nitrogen and 37°C water bath...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a bovine CFL2 gene adenovirus interference vector and a construction and identification method thereof.The bovine CFL2 gene adenovirus interference vector is characterized in that Ad-CFL2 shRNA-1 with a good interference effect is used for infecting bovine primary muscle cells, packaging and propagation of adenovirus are carried out, and by constructing the bovine CFL2 gene adenovirus interference vector, the bovine CFL2 gene adenovirus interference vector can be obtained. The purpose of continuously knocking down CFL2 gene expression in bovine muscle tissues and cells can be achieved, an effective technical support is provided for follow-up research on a molecular regulation mechanism between the CFL2 gene and muscle growth and development, and a new molecular marker and breeding thought can be further provided for genetic improvement and efficient breeding of local cattle in China.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bovine CFL2 gene adenovirus interference vector and its construction and identification method. Background technique [0002] Muscle is an important factor affecting the growth and development of animals, and it is also one of the important indicators to measure meat quality, and the growth and development of muscle is a very important quantitative trait, which is the result of the sequential expression and closure of a series of related genes, and its phenotype has special At the same time, the meat production capacity of animals is closely related to the number and growth of muscle cells. However, since the current breeding methods for livestock are still in the initial stage of transformation from traditional breeding to molecular breeding, it is of great guiding significance to study the regulation mechanism of muscle growth and development at the mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N15/113C12Q1/6851C12N15/11
CPCY02A50/30
Inventor 孙雨佳赵天奇马瑶瑶孙晓梅杨章平
Owner YANGZHOU UNIV
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