Lentivirus (Lentivirus) vector is gene therapy vectors, which is based on HIV-1 (human immunodeficiency virus type I). Compared to those general retroviral vectors, it has the ability to infect dividing cells and non-dividing cells. Lentiviral can produce the high-titer lentiviral, which can be used in shRNA expression.
This post will give customers a brief look at the construction of lentiviral vector in Creative Biogene.
Experimental procedures
1.Construction of a lentivirus overexpressing plasmid vector
Designing specific amplification primers at upstream and downstream, while the restriction sites are being introduced. with the help of high-fidelity KOD enzyme, the mutation rate of products in PCR is 0% within 3k length. The CDS (coding sequence) region of the target gene from template (CDNA plasmid or library) would be inserted to the T vector. The CDS region is cleaved by restriction endonuclease, and then cloned into lentivirus overexpressing plasmid vector.
2.Interference of lentivirus in the construction of plasmid vector
Synthesizing the neck ring structure of DNA which is corresponding to siRNA sequence, and then it is integrated into lentivirus plasmid vector.
3.Packaging and concentrated purification of lentiviral vectors
Packaging involves the preparation of competent virus from DNA vectors. Lentiviral vector production systems are totally depended on a 'split' system, in which the natural viral genome has been split into single helper plasmid constructs. This splitting of different viral elements into four separate vectors reduces the risk of creating a replication-capable virus with the help of adventitious recombination of the lentiviral genome.What's more, a vector with the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) is transiently transfected into human 293 cells. The virus containing supernatant are harvested and concentrated after a 48-hour incubation period. Finally, virus titer is detected by reporter (fluorescent) expression with a flow cytometer.
Experimental material Lentiviral vector, packaging cells and strain The virus packaging system is three plasmid systems, containing pspax2, pMD2G, pLVX-IRES-ZsGreen1/pLVX-shRNA2. The ZsGreen1 expression cassette on plasmid is capable of expressing the green fluorescent protein (GFP).
Bearer information Lentiviral cloning vector maps as follows:
This is the main experimental procedures for the construction of lentiviral vectors. Lentiviral vectors (Lentiviral vector) compared with retroviral vectors have a wider host range.
Related service: Lentivirus service