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Human colorectal adenocarcinoma cell TRPV1 gene knockout cell strain and application

A rectal adenocarcinoma and gene knockout technology, applied in the field of genetic engineering, can solve the problems of affecting experimental results, failure of antagonism, difficulty in controlling the dosage of antagonists, etc.

Pending Publication Date: 2020-08-04
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the antagonist Capsicum zopin is widely used, it has certain limitations
First, it is difficult to control the amount of antagonist used in the experiment, and the appropriate inhibitory concentration can only be found through experiments with different doses; second, the antagonist itself has certain toxic effects on experimental animals or cells, and if the amount is too much, it will cause The possibility of experiment failure; third, the metabolic rate of the antagonist in the test animal or in cells is different from that of capsaicin, and the metabolic rate is greater than that of capsaicin, which will lead to the failure of the antagonistic effect and affect the experimental results

Method used

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  • Human colorectal adenocarcinoma cell TRPV1 gene knockout cell strain and application
  • Human colorectal adenocarcinoma cell TRPV1 gene knockout cell strain and application
  • Human colorectal adenocarcinoma cell TRPV1 gene knockout cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of human colorectal adenocarcinoma cell line with TRPV1 gene knockout

[0030] 1. Experimental method

[0031] (1) Determination of the minimum total lethal concentration of purine toxin (Puro) on cells

[0032] Digest Caco-2 cells with trypsin to prepare Caco-2 cell suspension, count the Caco-2 cell suspension, inoculate 50,000 cells per well of 24-well plate, 500 μL per well, add Puro at different concentrations of 0, 0.5, 0.75, 1, 2, and 4 μg / mL was observed under the microscope after 2-3 days, and the lowest concentration that was completely lethal to the cells was selected as the drug screening concentration for subsequent experiments.

[0033] (2) Monoclonal growth verification test

[0034] Digest Caco-2 cells with trypsin to prepare Caco-2 cell suspension, take Caco-2 cell suspension, count and perform limiting dilution, so that the number of cells per well in the final 96-well plate is 1, and the diluted Cells at 37°C, 5% CO 2 Culture...

Embodiment 2

[0066] Example 2 Detection of protein expression level of TRPV1 gene knockout cell line

[0067] 1. Experimental method

[0068] Use the mixture of RIPA protein lysate and protein phosphatase inhibitor to lyse the cells, measure the protein concentration after centrifugation, take 20μg protein sample and mix with 5×SDS protein sample buffer, boil at 95°C for 10min, and use 12% separation gel SDS-PAGE protein electrophoresis, 80V for 40min, 100V for 30min; wet transfer at 200A for 1.5h; block with 5% skimmed milk in TBST solution at room temperature for 1.5 hours; anti-TRPV1 polyclonal antibody (Abcam) diluted 1:1000. GAPDH (Abcam ) was diluted 1:10000 and incubated at room temperature for 2 hours. Wash 3 times with TBST, 10 min each time.

[0069] 2. Experimental results

[0070] The expression of TRPV1 protein in the cells was detected. Such as Figure 7 As shown, the TRPV1 protein expression level of the knockout cell line was significantly lower than that of the wild c...

Embodiment 3

[0071] Example 3 Comparison of TRPV1 gene knockout cell line and wild cell proliferation rate

[0072] 1. Experimental method

[0073] The WT and KO cells in the logarithmic growth phase were digested into cell suspensions with digestive fluid, counted with a cell viability detection instrument, and the cell concentration was adjusted to 1.25×10 5 Viable cells / mL, then inoculated on 6-well cell plates, 2 mL per well, placed in a 37°C, 5% CO2 incubator for static culture, and digested and counted cells in 3 wells at 12h, 24h, 36h, and 48h after inoculation , draw the growth curves of the two kinds of cells, and observe whether TRPV1 gene knockout affects the growth efficiency of Caco-2 cells.

[0074] 2. Experimental results

[0075] In order to evaluate whether TRPV1 gene knockout has any effect on the growth performance of Caco-2 cells, the growth rates of the two kinds of cells were measured respectively. There was no significant difference in the growth rate between 0-48h ...

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Abstract

The invention discloses a human colorectal adenocarcinoma cell TRPV1 gene knockout cell strain and application. The cell strain is named as a human colorectal adenocarcinoma cell TRPV1 gene knockout cell strain CACO-2 KO, and was preserved in China Center for Type Culture Collection on December 25, 2019, and the preservation number is CCTCC NO. C202024. According to the invention, based on the CRISPR / cas9 technology, a plasmid vector constructed by using a pair of gRNAs represented by SEQ ID NO: 2-3 is adopted to electrically transfect a cell to be knocked out, and resistance and sequencing screening are performed to obtain the recombinant plasmid vector. The designed gRNA can accurately and efficiently knock out the TRPV1 gene, and a human colorectal adenocarcinoma cell line with the TRPV1 gene knocked out is effectively constructed. The establishment of the TRPV1 gene knockout cell line provides effective gene materials and gene tools for researching the action mechanism of capsaicine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a human colorectal adenocarcinoma TRPV1 gene knockout cell line and its application. Background technique [0002] The latest development of the CRISPR / Cas9 system, an efficient, programmable genome editing tool, has revolutionized basic scientific research and has proven to be a major scientific breakthrough. Technologies based on the CRISPR / Cas9 system provide researchers with new and powerful tools for creating accurate cellular and animal models of human disease. Unlike the protein-guided DNA cleavage used by TALENs and ZFs, CRISPR / Cas9 relies on a small RNA to recognize the knockout target, thereby introducing a site-specific double-strand DNA break, and its development makes gene editing much easier. CRISPR / Cas9 technology has been widely used in new animal models since its inception. Before that, the construction of knockout animal models was a ti...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/11C12N15/85C12N15/90C12R1/91
CPCC07K14/705C12N15/113C12N15/85C12N15/907C12N2310/20
Inventor 王远微
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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