Construction of TALENs Monoclonal Stable Cell Line
T7E1 activity analysis experiment is used to assay the activity of TALENs plasmid at AAVS1 site. Experiment shows that the plasmid can identify the TTTCTGTCACCAATCCT and CCCCTCCACCCCACAGT sequences. More
Summary: T7E1 activity analysis experiment is used to assay the activity of TALENs plasmid at AAVS1 site. Experiment shows that the plasmid can identify the TTTCTGTCACCAATCCT and CCCCTCCACCCCACAGT sequences. The cutting efficiency is about 10%. With the Donor plasmid, the exogenous gene can be inserted into the AAVS1 site efficiently. Stable cell line can be screened, which is completely different from knockout cell lines.
The TALENs plasmid cut from AAVS1 site and homologous plasmid are used to identify. Homologous recombination repair mechanism designates the expression (CMV-EGFP-IRES-Puro-poly A) to the AAVS1 site on chromosome 19. Then monoclonal selection techniques is used to obtain stable cell lines which can express the EGFP and Puromycin resistance genes. The stable cell line can be effectively integrated with other exogenous genes by cre/loxp technology.
The traditional stable strain screening method needs to screen the target cells after transient transfection, and finally obtain the stable cell line from single cell. This method has low positive rate, long cycle and heavy workload. At the same time, the position of the integrated exogenous genes in the stable strains is not determined, which is easy to cause interference. The method of integrating the slow virus into the host genome has overcome the disadvantages of traditional methods, and can obtain high efficiency stable cell lines in a short time. But the obtained stable strains have mixed sites. The TALENs technique can be used to integrate the exogenous gene into the recognized safety site AAVS1, which can lay a good foundation for the study of gene function.
To obtain 293T cell lines with TALENs technology and EGFP/Puromycin resistance genes.
1.Cells are plated
The 293T cells are inoculated into 6 well plates with the fusion of 70%.
Transfecting cells in 16h according to the transfection reagent (4μg pTALEN-AAVS1-F, pTALEN-AAVS1-R, Fixed template plasmid (1:1:1), 10μg lipofectamine, 2000).
3.Stable strain selection
After 72h, 2μg/ L puromycin is added. Fresh 2μg/ L puromycin replaces every 2-3 days;
After 3-4 days, put all the single cell on 96 well plate;
Selection of fluorescent cells that continue to expand after the amplification;
Extraction of genomic DNA, PCR product for sequencing.
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