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Gene knockout carrier and gene knockout method of NLRP1 gene of MH7A cell

A gene knockout and vector technology, applied in the field of molecular biology, can solve problems such as not very clear, and achieve the effect of promoting research and promoting pathogenesis

Inactive Publication Date: 2017-05-31
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The etiology of RA is still not very clear, but studies in recent years have shown that its pathogenesis may be related to nucleotide-binding oligomerization domain-like receptor protein 1 (nucleotide-binding, leucine-rich repeat pyrin domaincontaining protein 1, NLRP1)

Method used

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  • Gene knockout carrier and gene knockout method of NLRP1 gene of MH7A cell
  • Gene knockout carrier and gene knockout method of NLRP1 gene of MH7A cell
  • Gene knockout carrier and gene knockout method of NLRP1 gene of MH7A cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 pre-experimental operation

[0054] 1. Monoclonal growth verification test.

[0055] Limiting dilution: Make MH7A cells into a cell suspension and count the cells, then take a certain amount of cell suspension for dilution, so that the number of cells in each well of the final 96-well plate is 1 or 5, (100 μl per well) after confirming that the dilution is accurate , will be diluted at 37°C, 5% CO 2 Static culture in the incubator; after 7 to 10 days, under the microscope, it was found that monoclones could be formed, and the cells had a tendency to proliferate and could form cell clusters.

[0056] 2. Explore the electroporation conditions of MH7A cells

[0057] After the MH7A cells were made into a uniform single-cell suspension, take a part of it and mix it evenly for platelet trypan blue staining and counting (the viable cell rate > 95% can be electroporated), and calculate the concentration of the cell suspension; take 1 × 10 7 Suspend the cells in ...

Embodiment 2

[0060] The construction method of embodiment 2 gene knockout vector

[0061] In this embodiment, a method for constructing a gene knockout vector for knocking out the NLRP1 gene is provided, comprising the following steps:

[0062] 1. Target site design and synthesis

[0063] 1) Design CRISPR-Cas9 knockout target sites

[0064] First, a pair of oligo DNA of about 20bp needs to be designed in the target DNA region, and designed through the following online tools:

[0065] CRISPR Design at MIT: http: / / crispr.mit.edu /

[0066] The common CDS region of the five transcripts of the NLRP1 gene was selected, and the first exon of the common CDS region was found for target site design. It is best to input only one exon at a time to avoid the Guide sequence spanning introns.

[0067]

[0068] Among them, bases in capital letters represent exons, and bases marked in bold and underlined represent selected target sequences.

[0069] The NCBI scoring of the target sequence is as fol...

Embodiment 3

[0093] In this embodiment, a method for knocking out the NLRP1 gene in MH7A cells is provided.

[0094] 1. Transform the gene knockout vector obtained in Example 2 into G10Competent Cell:

[0095] Take 1 tube of G10Competent Cell from the -80°C refrigerator and thaw on ice.

[0096] After melting, add 10 μl of the ligation product, flick to mix, and incubate on ice for 30 minutes.

[0097] Shock in a water bath at 42°C for 60 sec, quickly take it out and place it on ice to cool for 2-3 min.

[0098] Add 800 μl of anti-SOC-free liquid medium to the tube, and resume culturing on a shaker (37° C. / 160 rpm) for 45 minutes.

[0099] Centrifuge at 4500 rpm for 5 min, discard 800 μl of supernatant, suspend the pellet in the remaining 100 μl of supernatant, spread evenly on the screening plate containing Kan resistance, and incubate overnight.

[0100] 2. Screen positive recombinants

[0101] On the second day of transfection, use the upstream primer VSP primer and the respective d...

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Abstract

The invention relates to the molecular biology field and particularly relates to a gene knockout carrier, a construction method and application thereof and a gene knockout method of an NLRP1 gene of an MH7A cell. A CRISPR-Cas9 gene knockout system is utilized for carrying out CRISPR targeting sequence design by taking NLRP1 as a target gene so as to prepare a knockout carrier aiming at the NLRP1 gene, and the knockout carrier is utilized for transfecting the MH7A cell, so that the NLRP1 gene in the MH7A cell can be efficiently knocked out; and therefore, a research platform for rheumatoid arthritis is effectively built, so that the research of the pathogenesis of the rheumatoid arthritis is greatly promoted, and the researches of the interaction of NLRP1 inflammasomes and various inflammatory factors and the disease related molecular mechanisms of the rheumatoid arthritis and meningitis can be promoted.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a gene knockout vector and its construction method and application, and a method for knocking out the NLRP1 gene of MH7A cells. Background technique [0002] Rheumatoid Arthritis (Rheumatoid Arthritis, RA) is a chronic autoimmune disease characterized mainly by synovitis of the joints. The incidence of RA in my country is about 0.3% to 0.6%. The incidence rate is high, the disability rate is high, and severe endanger human health. [0003] The etiology of RA is still not very clear, but studies in recent years have shown that its pathogenesis may be related to nucleotide-binding oligomerization domain-like receptor protein 1 (nucleotide-binding, leucine-rich repeat pyrin domaincontaining protein 1, NLRP1). The innate immune system recognizes invading microorganisms and danger signals in the body through specific pattern-recognition receptors (PRRs). PRR is mainly divided...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N5/10
CPCC12N15/85C07K14/705C12N5/0656C12N15/66C12N2510/00C12N2800/107C12N2800/80C12N2810/10
Inventor 钟兵方勇飞王勇
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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