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Method for improving transfection efficiency of B16F10 cells based on CRISPR-Cas9 technology

A B16F10, transfection efficiency technology, applied in the field of improving B16F10 cell transfection efficiency based on CRISPR-Cas9 technology, can solve problems such as low cell transfection efficiency, limited cell transfection efficiency, and limited gRNA activity verification detection, etc. The effect of improving transfection efficiency

Inactive Publication Date: 2018-09-21
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, many biological function studies and multi-drug treatments involving gene therapy are limited by the transfection efficiency of cells
[0003] B16F10 cells are a mouse melanoma cell line that is easy to culture; it is currently used to verify the activity of Cas9gRNA in vitro in mice, but the transfection efficiency of the cells is not high at present, and the lipo2000 lipotransfer efficiency of commonly used lipo2000 is about 10%, which greatly limits the follow-up Assays for gRNA Activity Validation

Method used

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  • Method for improving transfection efficiency of B16F10 cells based on CRISPR-Cas9 technology
  • Method for improving transfection efficiency of B16F10 cells based on CRISPR-Cas9 technology
  • Method for improving transfection efficiency of B16F10 cells based on CRISPR-Cas9 technology

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Embodiment 1

[0039] 1. Targeted knockout SQSTM1 lentiviral plasmid construction

[0040] 1.1 Synthesis of sgRNA oligonucleotide chain

[0041] Use the CRISPR online design tool (http: / / crispr.mit.edu / ) to design a 20bp sgRNA on the second exon of SQSTM1 according to the scoring system, and verify that there is no non-specific gene by BLAST. Add ACCG to the 5'end of the coding strand template, and add AAAC to the 3'end of the non-coding strand template, which is complementary to the sticky end formed by BsmBI digestion. Design a pair of CRISPR oligonucleotide strands, see Table 1, Table 1 SQSTM1 targeting site Dot and sgRNA oligonucleotide sequence

[0042]

[0043] 1.2 Vector construction

[0044] 1.2.1 Use BsmBI to digest 2μg pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9 plasmid, 2h, 37℃, digestion system

[0045]

[0046] 1.2.2 Purify the digested plasmid product using GENRY gel recovery kit, and operate according to the instructions

[0047] 1.2.3 Phosphorylation and annealing of sgRNA, enzyme...

Embodiment 2

[0063] 1. Transfection of SQSTM1 knockout cell line

[0064] 1.1 The SQSTM1 knockout cell line and the control empty cell line (B16F10) were plated in 1 well of a 6-well plate respectively;

[0065] 1.2 Arrange for transfection when the cell confluence reaches 80% on the second day; prepare DNA and transfection reagents during transfection: the ratio of each well of the 6-well plate plasmid is plasmid pEGFP-N1 ( figure 2 ): Transfection reagent (lipofectmine 2000) = 5ug: 5ul; Incubate the diluted DNA and transfection reagent for 5 min at room temperature; mix the diluted DNA and transfection reagent and incubate at room temperature for 20 min;

[0066] Discard the supernatant of the medium in the well plate, suck up the remaining liquid, add fresh medium without double antibodies; add the mixed plasmid and transfection reagent dropwise to the well; after 6 hours of transfection, replace with fresh Complete medium; 48 hours after transfection, under the microscope, the transfection e...

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Abstract

The invention discloses a method for improving the transfection efficiency of B16F10 cells based on CRISPR-Cas9 technology. The method comprises the following steps of: firstly, obtaining an sgRNA sequence specifically targeted to a second exon of the SQSTM1 gene; then constructing the sgRNA of the SQSTM1 gene to the lentiviral vector system, and the vector expresses the Cas9 protein; and finally,transfecting the CRISPR / Cas9 lentiviral vector containing the sgRNA into B16F10 cells, and obtaining a knock-out cell strain of the SQSTM1 after the drug is screened. The transfection efficiency of the B16F10 knockout strain of the obtained SQSTM1 is significantly higher than that of the unknocked-out cells. The method has the advantages of simple operation steps, good sgRNA targeting property and high cutting efficiency to the SQSTM1 gene; and the transfection efficiency of the B16F10 cells can be obviously improved, and a better cell tool is provided for the basic research.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a method for improving the transfection efficiency of B16F10 cells based on CRISPR-Cas9 technology. Background technique [0002] Transfection is a technology that introduces exogenous nucleic acids (including plasmids, siRNA, etc.) into eukaryotic cells and exerts their functions. Transfection is currently widely used in biological research. However, many biological function studies and multiple drug treatments involving gene therapy are limited by the transfection efficiency of cells. [0003] B16F10 cells are a mouse melanoma cell line that is easy to culture; they are currently used to verify Cas9gRNA activity in vitro in mice. However, the current transfection efficiency of this cell is not high. The commonly used lipo2000 lipotransfection efficiency is about 10%, which greatly limits subsequent follow-up. Assay for verification of gRNA activity. [0004...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10
CPCC07K14/47C12N15/113C12N15/86C12N2310/10C12N2740/15043
Inventor 杨佳丽杨兴林潘讴东
Owner OBIO TECH SHANGHAI CORP LTD
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