Cell model obtained after targeted knockout of rabbit bone morphogenetic protein-2 (BMP2) gene based on CRISPR/Cas9 and application thereof

A cell model and gene technology, applied in the fields of molecular biology and biomedicine, can solve the problem that the mechanism of tumor action is not completely clear, and achieve good stability and reliability, low cost, and simple preparation methods

Inactive Publication Date: 2016-10-26
QINGDAO JIAOZHOU CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the osteogenesis mechanism of BMP-2 has been relatively clear, and its close relationship with the corresponding TGF-β ligand and receptor has drawn more and more attention to the relationship between BMP-2 and tumor occurrence and development, but its The mechanism of action in tumors has not been fully elucidated

Method used

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  • Cell model obtained after targeted knockout of rabbit bone morphogenetic protein-2 (BMP2) gene based on CRISPR/Cas9 and application thereof
  • Cell model obtained after targeted knockout of rabbit bone morphogenetic protein-2 (BMP2) gene based on CRISPR/Cas9 and application thereof
  • Cell model obtained after targeted knockout of rabbit bone morphogenetic protein-2 (BMP2) gene based on CRISPR/Cas9 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 gRNA synthesis and vector construction targeting the rabbit BMP2 gene

[0017] 1. Selection and design of gRNA targeting rabbit BMP2 gene

[0018] Find the sequence of the rabbit BMP2 gene in Genebank, and design potential target sites in the first exon region of the rabbit BMP2 gene. Through the online design tool (http: / / crispr.mit.edu / ) and gRNA design principles, evaluate the high-scoring target site design gRNA on the rabbit BMP2 gene sequence, the target site sequence is SEQ ID NO.1-SEQ ID NO.3, and design the corresponding oligonucleotide.

[0019] 2. Synthesis of gRNA oligonucleotide sequence targeting rabbit BMP2 gene and construction of eukaryotic expression vector

[0020] The pSpCas9(BB)-2A-GFP(PX458) plasmid (AddgeneplasmidID: 48138, hereinafter referred to as pSpCas9(BB)) was digested with BbSI, and after 1 hour in 37°C water bath, 1% agarose electrophoresis was performed to recover the digested product ( TAKARA gel recovery kit).

[0021] Th...

Embodiment 2

[0033] Example 2 Lipofection of Rabbit Bone Marrow Mesenchymal Stem Cells to Establish a BMP2 Gene Knockout Cell Model

[0034] Three days before transfection, rabbit bone marrow mesenchymal stem cells (BMSCs, purchased from Saiye Biotechnology Co., Ltd.) were revived, and the cells were placed in a culture bottle with complete medium for rabbit bone marrow mesenchymal stem cells, at 37°C, 5% Cultured in a CO2 incubator, the day before transfection, subculture the recovered cells.

[0035] Aspirate the medium in the T75 bottle for culturing BMSCs cells, add 2 mL of 0.25% trypsin taken out of a 4°C refrigerator to make it evenly cover the bottom of the bottle, place it in a 37°C incubator for 3-5min, take it out, and shake it to find that the cells are in the The bottom is detached, shake it all down, add 3mL of rabbit bone marrow mesenchymal stem cell complete medium preheated in a 37℃ water bath, blow and beat with a 10mL pipette, blow and beat 6-8 times, leaving no dead spac...

Embodiment 3

[0044] Example 3 Cloning of PCR products and sending samples for sequencing detection

[0045] According to the method of Example 2, the cells transfected with the highly active SEQ ID NO.2 target construction vector were diluted and cultured to obtain cell clones. And identify cell clones.

[0046] Extract cell DNA and perform target site PCR. The PCR product is purified with TAKARA kit and then connected to the PMD18-T vector. The connection system is:

[0047]

[0048] Ligation was carried out at 16°C for 2 hours. Take competent cells DH5α, place in ice to melt for 5 minutes, add 10ul of ligation product and blow evenly, place in ice for 20 minutes. Heat shock at 42°C for 90s, quickly transfer to an ice bath and let stand for 3min, add 500ul LB liquid medium, place in a shaker, 180rpm at 37°C for 1h. Take 100 ul of the bacterial solution and evenly spread it on LB solid medium (containing 1 / 1000 AMP), and cultivate overnight at 37°C.

[0049] Pick 3 single colonies, ...

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Abstract

The invention relates to a cell model obtained after targeted knockout of a rabbit BMP2 gene based on CRISPR/Cas9 and application thereof, belonging to the technical field of molecular biology and biomedicine. According to the invention corresponding oligos are synthesized for three targeting sites of the rabbit BMP2 gene on the design principles of CRISPR/Cas9 and are constructed on px458 vectors; and a CRISPR/Cas9 system is constructed directed at the three targeting sites in rabbit mesenchymal stem cells, and the CRISPR/Cas9 system can effectively knock out the rabbit BMP2 gene, is easy to operate and has high rabbit BMP2 gene knockout efficiency. The cell model disclosed in the invention can greatly promote research related to the functions and signaling pathways of the BMP2 gene.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine, and in particular relates to a CRISPR / Cas9-based targeted knockout rabbit bone morphogenetic protein 2 (BMP2) gene cell model and its application. Background technique [0002] The CRISPR / Cas system is developed from the adaptive immune system of bacteria and archaea against foreign viruses or plasmids, including three different types, of which the Type II CRISPR / Cas system has only one subunit of the DNA endonuclease Cas9 , the structure is the simplest, so it is the most widely used. In addition to the Cas9 protein, the system also includes two short CRISPR RNAs (crRNAs) and trans-activatingcrRNAs (tracrRNA). The mature crRNA-tracrRNA complex can guide the Cas9 protein to the target sequence through complementary base pairing, and specifically cut the DNA double strand near the PAM (protospace radjacent motif) to form a DSB (double strand break). DSB can be repaired ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N15/85
CPCC07K14/51C12N15/85C12N2800/107C12N2810/10
Inventor 申友亮朱同娥张靖靖李修敏
Owner QINGDAO JIAOZHOU CENT HOSPITAL
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