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
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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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