Non-viral episomal vector capable of controlling deletion and construction method thereof
A vector and sequence technology, applied in the field of genetic engineering, can solve problems such as increasing the risk of iPS transforming into cancer cells, and achieve the effect of high efficiency and simple operation process
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Embodiment 1
[0024] The construction of embodiment 1 episomal vector
[0025] The vector construction steps are as follows, and the construction process is as follows figure 1 ;
[0026] 1. NheI and AgeI double-digest the pEPI-EGFP vector, insert the first loxp sequence, and obtain the intermediate vector 1,
[0027] 2. SalI and BmHI double-digest the intermediate vector 1, insert the second loxp sequence in the same direction to obtain the intermediate vector 2,
[0028] 3. PciI and NheI double digest the intermediate vector 2, delete the CMV promoter, insert the human EF1α promoter to replace the CMV promoter, and obtain the intermediate vector 3,
[0029] 4. Double digestion with BsaI and MluI, delete the neomycin resistance gene of the intermediate vector 2, insert the ampicillin resistance gene, and obtain the intermediate vector 4,
[0030] 5. AscI and SpeI double digestion pPB-CAG-TKiNeo vector to obtain the target fragment, insert AscI and SpeI double digestion intermediate vect...
Embodiment 2
[0031] Example 2 Cell Transfection and Controlled Deletion of Episomal Vectors
[0032] 1. Use the endotoxin-free plasmid extraction kit to extract the pEPI-EF1α-EGFP-CAG-TKiNeo plasmid, dissolve it in ultrapure water until the concentration reaches 1ug / ul, and carefully pipette 3ug of the plasmid and 5×10 6 After the cells were fully mixed, they were electrotransfected. After electrotransfection, the cells were plated on five 10cm cell culture dishes, and G418 was added to a concentration of 500ug / ml after 2 days of culture. After 10 days of screening, cell clones expressing green fluorescence could be seen, such as image 3 .
[0033] Because the expression of EGFP gene enhances green fluorescent protein, the cell clones can express green fluorescence, which proves that the pEPI-EF1α-EGFP-CAG-TKiNeo plasmid has been successfully transfected into the cells.
[0034] 2. Add TAT-Cre protein to a concentration of 400ng / ml and incubate for 24 hours, then add 0.2umol / Lganc to scr...
Embodiment 3
[0036] Example 3 Construction of Deletable Episomal Vectors for Inducing iPS
[0037] AscI digested pEPI-EF1α-EGFP-CAG-TkiNeo, recovered the linear vector from the gel, treated the linear vector with alkaline phosphatase, removed the 5’ phosphate of the linear vector, and used pSTEM as the template.
[0038] Primer F: aatGGCGCGCCTCGTGAGGCTCCGGTGCC;
[0039] With primer R: aatGGCGCGCCCTTACAATTTACGCCTTAAGATACATTG (the underlined part is the AscI restriction site, and the lowercase is the protective base that needs to be added for direct digestion of the PCR product), amplify to the target fragment, link with the linearized vector after AscI digestion, and transform, The target clone was obtained by screening, and the cloning vector map is as follows: Figure 4 shown.
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