SgRNA for targeted knockout of RPSA gene and construction method of RPSA gene knockout cell line

A cell line and gene technology, applied in the field of genetic engineering, can solve problems such as lame production performance, low lethality, and high pathogenicity, and achieve the effects of accurate targeting, increasing antigen expression, and promoting replication

Active Publication Date: 2020-10-30
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Foot-and-mouth disease is highly pathogenic, but the lethality rate is low. Long-term illness of animals will cause lameness and decline in production performance, which has a very negative economic impact on farmers

Method used

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  • SgRNA for targeted knockout of RPSA gene and construction method of RPSA gene knockout cell line
  • SgRNA for targeted knockout of RPSA gene and construction method of RPSA gene knockout cell line
  • SgRNA for targeted knockout of RPSA gene and construction method of RPSA gene knockout cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Design of sgRNA targeting RPSA gene

[0073] Use the NCBI database to query the RPSA gene sequence, and find the whole genome of the golden hamster (GenBank accession number: NW_004801729), locate the first exon segment of RPSA in the overlapping region of different transcripts in the genome, and use it for target design.

[0074] According to the CRISPR / Cas9 design principle, log in to the CRISPR online design website http: / / crispr-era.stanford.edu / index.jsp to design sgRNA, select 4 pairs of 20bp sgRNA fragments according to the score, and name them respectively: RPSA-sg RNAsp1, RPSA-sgRNAsp2, RPSA-sgRNAsp3, RPSA-sgRNAsp4: add CACC sticky end to the 5' end of the forward sequence of the sgRNA fragment, and add AAAC sticky end to the 5' end of the reverse sequence, as sgRNA oligos targeting RPSA gene polynucleotide (sgRNA-oligo). The sgRNA-oligo was synthesized by Jinweizhi Biotechnology Co., Ltd., and the detailed sequence is shown in Table 1.

[0075] Table...

Embodiment 2

[0078] Example 2 Construction of sgRNA recombinant plasmid PX330-sgRNA

[0079] Obtaining double-stranded sgRNA-oligo: dilute the sgRNA-oligo synthesized in Example 1 to 100 μmol / L, and prepare a total of 10 μl reaction system: upstream primer 2.5, μl; downstream primer, 2.5 μl; ddH 2 O, 4 μl; 10×TaqBuffer, 1 μl. Reaction program: Annealing treatment according to 0.3℃ / s gradient cooling, 95℃, 3min; 95℃, 1min; 85℃, 1min; 75℃, 1min; 65℃, 1min; 55℃, 1min; 45℃, 1min; 35℃ ℃, 1min; 25℃, 1min; 16℃, 1h; anneal the upstream and downstream primers to form a double-stranded sgRNA-oligo.

[0080] Enzyme digestion of PX330 vector plasmid: digest PX330 vector with BBSI restriction endonuclease, and prepare 20 μl of enzyme digestion system as follows: PX330 vector, 5 μl; BBSI, 1 μl; 10×Buffer, 2 μl; ddH 2 O, 12 μl. Place at 37°C for 2h for enzyme digestion. Afterwards, nucleic acid electrophoresis was carried out, and the DNA purification and recovery kit of Promega was used to purify an...

Embodiment 3

[0083] Example 3 Cell Transfection

[0084] Resuscitate BHK21 cells in T25 cell flasks before transfection, and culture them in DMEM medium containing 10% FBS and 1% double antibody. In the cell six-well plate, when the degree of cell fusion reached 70% to 80%, the recombinant plasmids successfully constructed in Example 2 (PX330-RPSA-sgRNA1, PX330-RPSA-sgRNA2, PX330-RPSA-sgRNA3, PX330- RPSA-sgRNA4) 2 μg and Lipofectamine3000, 4 μl (according to the ratio of 1 μg: 2 μL) were added to 50 μl of Opti-MEM, and the two were mixed after standing still for 5 minutes. The four liposome-plasmid DNA mixtures were allowed to stand for 15 minutes and then directly added to the cell culture medium. Return the cells to 37°C, 5% CO 2 Cultivate in the incubator for 48h.

[0085] 1. Cell DNA extraction and SURVEYOR experiment:

[0086] Extract the total DNA of BHK21 cells 48 hours after transfection with the PX330-sgRNA recombinant plasmid according to the operation instructions of the mic...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to SgRNA for targeted knockout of an RPSA gene and a construction method of an RPSA gene knockout cell line. Accordingto the sgRNA of a specific targeted RPSA gene, the sgRNA can specifically target an RPSA gene, the complete knockout of the RPSA gene in a host cell is realized by applying a CRISPR-Cas9 technology, and the knockout efficiency is high; the invention also provides a method for transfecting the sgRNA into a host cell by virtue of a CRISPR-Cas9 technology to construct an RPSA gene knockout cell line.Knocking out the RPSA gene in the host cell not only can promote replication of the FMDV virus, but also can be used for improving the production quantity and antigen expression quantity of the FMDVvaccine; in addition, it is accidentally found that by knocking out the RPSA gene in the host cell, replication of the Seneca valley virus in the cell can be remarkably inhibited; a research tool anda material are provided for further researching a molecular mechanism of the RPSA gene for regulating and controlling pathogenic microorganism replication in cells.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing sgRNA targeting knockout of RPSA gene and RPSA gene knockout cell line. Background technique [0002] Genome manipulation technology is a cutting-edge technology developed based on genome and genetic information technology to achieve precise editing of specific genes or genome target sites through artificial design. It has become a research hotspot in the fields of biomedicine, agricultural animal breeding, and model animals. The emergence of genome editing (Genome editing) technology for precise modification and directional editing of the genome by artificially causing specific base insertions, deletions, or substitutions in the genome has made new progress in various fields of life sciences and has been widely used. in basic research and clinical treatment. The original gene editing technology is to introduce exogenous DNA into cells by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N5/10C12N7/00C12R1/91C12R1/93
CPCC12N15/113C12N15/85C12N15/907C12N5/0686C12N7/00C12N2310/20C12N2800/107C12N2510/00C12N2770/32151
Inventor 郑海学朱紫祥张向乐高利利杨帆曹伟军王聪聪刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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