Lmna gene knockout cell line constructed based on CRISPR/Cas9 technology

A gene knockout and cell line technology, applied in the biological field, can solve problems such as insufficient research and lack of gene knockout cell lines

Inactive Publication Date: 2021-08-20
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on the relationship between LMNA gene and disease is still insufficient, and there is a lack of corresponding gene knockout cell lines, so it is necessary to provide a cell model of LMNA gene knockout, so as to provide the necessary tools for scientific research

Method used

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  • Lmna gene knockout cell line constructed based on CRISPR/Cas9 technology
  • Lmna gene knockout cell line constructed based on CRISPR/Cas9 technology
  • Lmna gene knockout cell line constructed based on CRISPR/Cas9 technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Construction of LMNA gene knockout 293T cell line

[0014] (a) Design of gRNA

[0015] to be knocked out LMNA Based on the complete sequence of the gene, the inventors designed two pairs of gRNA primers, which are:

[0016] gRNA7

[0017] CACCGGCACGCAGCTCCTGGAAGGGT (SEQ ID NO. 1),

[0018] AAACACCCTTCCAGGAGCTGCGTGCC (SEQ ID NO. 2), and

[0019] gRNA8

[0020] CACCGGCGCCGTCATGAGACCCGAC (SEQ ID NO. 3),

[0021] AAACGTCGGGTCTCATGACGGCGCC (SEQ ID NO. 4).

[0022] (b) Vector construction

[0023] (b1) Digestion of empty plasmid and gel purification Digest 1ug of plasmid with restriction endonuclease BbsI, 37°C, 30min:

[0024] BBSI 1ul PX459 1ug 10xBbSI buffer 5ul Sterilized water Make up to 50ul

[0025] Plasmids digested with the QIAquick Gel Extraction Kit were gel purified and eluted in EB.

[0026] (b2) Phosphorylation and annealing gRNA reaction system:

[0027] gRNA-F 1ul gRNA-R 1ul 10x...

Embodiment 2

[0052] Construction of the HePG2 cell line of embodiment 2 LMNA gene knockout

[0053] (a) gRNA primer design

[0054] Based on the complete sequence of the lmna gene to be knocked out, the inventors designed two pairs of gRNA primers, which are:

[0055] gRNA 5

[0056] CACCGGTTCCGCCAGCAGCCGCCGGC (SEQ ID NO. 7),

[0057] AAACGCCGGCGGCTGCTGGCGGAACC (SEQ ID NO. 8); and

[0058] gRNA 6

[0059] CACCGGAGCGGGAGATGGCCGAGATG (SEQ ID NO. 9),

[0060] AAACCATCTCGGCCATCTCCCGCTCC (SEQ ID NO. 10).

[0061] (b) Vector construction

[0062] (b1) Digestion of empty plasmid and gel purification Digest 1ug of plasmid with restriction endonuclease BbsI, 37°C, 30min:

[0063] BBSI 1ul PX459 1ug 10xBbSI buffer 5ul Sterilized water Make up to 50ul

[0064] Plasmids digested with the QIAquick Gel Extraction Kit were gel purified and eluted in EB.

[0065] (b2) Phosphorylation and annealing gRNA reaction system:

[0066] gRNA-F 1ul gR...

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Abstract

The invention discloses a LMNA gene knockout cell line, the cell line is a 293T cell line or a HePG2 tumor cell line. The cell line is based on the crispr-cas9 technology and utilizes two pairs of gRNAs shown in SEQ ID NO.1-4 or two pairs of gRNAs shown in SEQ ID NO.7-10 to construct plasmid vectors to transfect cells to be knocked out. Resistance screened. The LMNA gene knockout cell line can be used for screening cell models for disease intervention drugs such as dilated heart disease, lipodystrophy syndrome, progeria syndrome and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LMNA gene knockout cell line constructed based on CRISPR / Cas9 technology and a construction method thereof. Background technique [0002] The CRISPR / Cas9 genome-directed editing technology developed in recent years can achieve specific and precise knockout of the genome. Genome-directed editing techniques can result in deletions, insertions, or substitutions of targeted loci. Following ZFN and TALEN technologies, the CRISPR / Cas9 system has rapidly developed into the 3rd generation genome editing technology. The CRISPR / Cas9 system is transformed from the CRISPR / Cas system II. Compared with ZFN and TALEN technologies, the CRISPR / Cas9 system is very simple in design, synthesis and screening, and it is easy to operate, low in cost, short in construction period, and can edit multiple genes at the same time, doubling the improvement of gene Editing efficiency. [0003] The LMNA gene ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N5/10C12N15/85C12R1/91
CPCC12N5/0693C12N15/85C12N15/907C12N2510/00C12N2800/80C12N2810/10
Inventor 舒伟刘恒杨晓波李东明李福记朱兰玉
Owner GUANGXI MEDICAL UNIVERSITY
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