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sgRNA for targeted knockout of FRZB gene, porcine embryo fibroblast cell line for knocking out FRZB gene and applications of fibroblast cell line

A porcine fibroblast and cell line technology, applied in the field of genetic engineering, can solve the problem of no gene knockout cell line, etc., and achieve the effect of large application research value, many germline mutations, and easy frameshift mutation

Active Publication Date: 2020-11-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the function of porcine FRZB gene, and there is no related gene knockout cell line

Method used

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  • sgRNA for targeted knockout of FRZB gene, porcine embryo fibroblast cell line for knocking out FRZB gene and applications of fibroblast cell line
  • sgRNA for targeted knockout of FRZB gene, porcine embryo fibroblast cell line for knocking out FRZB gene and applications of fibroblast cell line
  • sgRNA for targeted knockout of FRZB gene, porcine embryo fibroblast cell line for knocking out FRZB gene and applications of fibroblast cell line

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Construction and detection of CRISPR / Cas9 targeting vector targeting FRZB gene

[0043] 1. Sequence design of FRZB gene sgRNA

[0044]Download the porcine FRZB gene sequence (accession number NC_010457.5) from the NCBI website, use the website http: / / crispr.mit.edu / to design knockout target sites on the first exon of the gene, and select 10 PCR sequencing of the sgRNAs showed that the nesting peaks were not obvious, and the editing efficiency was low. Four sgRNAs with relatively high nesting peaks were selected and combined in pairs, and the editing efficiency was improved. Finally, the one with the highest sequencing peaks and the highest editing efficiency was selected. A set of double sgRNA, the two target sequences are FRZB-sgRNA1: CAGCACTCAGGCCAACGCCA (shown in SEQ ID NO: 1), FRZB-sgRNA2: CTCGTGGCCGGAAAGCCTGGC (shown in SEQ ID NO: 4). According to the BbsI restriction endonuclease, design restriction endonuclease sites at both ends of the sgRNA, add CAC...

Embodiment 2

[0067] Example 2 Construction and Genome Identification of FRZB Gene Knockout Pig Fibroblast Cell Line

[0068] 1. Screening of positive monoclonal cells

[0069] (1) When the porcine fibroblasts grow to a confluence of 70% to 90%, prepare a mixture containing 150 μL electroporation solution, 5 μg pX330-EGFP-FRZB-sgRNA1 and 5 μg pX330-EGFP-FRZB-sgRNA2 expression vector, use Lonza The T-024 program of the electroporation instrument was used for electroporation.

[0070] (2) After 6 hours of electroporation, replace with growth medium containing 10% fetal bovine serum. After 48 hours of transfection and electroporation, a large number of successfully transfected green fluorescent positive cells can be seen under the microscope (such as Figure 4 shown), the positive single clones with green fluorescence were sorted out by flow cytometry, and injected into a 96-well plate with preheated medium at the amount of 1 cell per well, and replenished every 3 to 4 days. Add the culture...

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Abstract

The invention relates to the technical field of genetic engineering, and especially relates to a sgRNA for targeted knockout of a FRZB gene, a porcine embryo fibroblast cell line for knocking out theFRZB gene and applications of the fibroblast cell line. The provided double sgRNA can cut two loci on the first exon of the FRZB gene, then after the sgRNA is constructed into an expression vector, then porcine embryo fibroblast cells are transfected through an optimized electro-transformation system, and a flow cytometer is used to sort positive monoclonal cells with high purity, so that the FRZBknocked-out cell line can be obtained through genome identification and screening. Thus, the problems of low transfection efficiency, low targeting efficiency and low monoclonal purity during the construction of knockout cell lines can be solved; and simple operation can be realized, and the edited cell line with longer fragment deletion can be obtained. The related cell line can be used as the donor of nuclear transfer of somatic cells for the preparation of transgenic swine, so that good research tools can be provided for further exploring the biological functions of the FRZB gene.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a double sgRNA targeted to knock out the FRZB gene and a porcine fibroblast cell line for knocking out the FRZB gene and applications thereof. Background technique [0002] Gene knockout is an effective means to study gene function and genetic improvement. CRISPR / Cas9 is currently the most commonly used gene editing system, widely used in the research of gene function and transgenic animal preparation. The main principle is to guide the Cas9 protein to the genomic region complementary to its sequence through sgRNA, so that the Cas9 protein binds to the target genome sequence, and performs genome cutting to cause DNA double-strand breaks, usually through non-homologous end repair. Insertion or deletion of one or several bases can be generated to achieve the purpose of gene function knockout. However, because the method for predicting the efficiency of sgRNA is not pe...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N15/85C12N5/10C12N15/11C12Q1/6888C12Q1/02A01K67/027C12R1/91
CPCC12N15/113C12N15/907C12N15/8509C12N5/0656A01K67/0276G01N33/5005C07K14/4703C07K14/475C12Q1/6888C12N2310/20C12N2510/00A01K2217/075A01K2227/108A01K2267/03
Inventor 张博付玉张盼商鹏张然张浩
Owner CHINA AGRI UNIV
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