LMNA gene knocked-out cell line constructed on basis of CRISPR/Cas9 technology

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

Inactive Publication Date: 2018-08-17
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 knocked-out cell line constructed on basis of CRISPR/Cas9 technology
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  • LMNA gene knocked-out cell line constructed on basis of 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

gR...

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-...

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Abstract

The invention discloses an LMNA gene knocked-out cell line. The cell line is a 293T cell line or a HePG2 tumor cell line. Cells to be knocked out are transfected by plasmid vectors constructed by twopairs of gRNA as shown in SEQ ID NO. 1-4 or two pairs of gRNA as shown in SEQ ID NO. 7-10 on the basis of a crispr-cas9 technology, and then are subjected to resistance screening to obtain the cell line. The LMNA gene knocked-out cell line can be used for intervention drug screening cell models for diseases such as dilated cardiomyopathy, fat metabolic disorder syndrome and premature aging syndromes.

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 Applications(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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