Multiple platforms of a HIV-2 derived lentiviral vector for expanded utility.

Using the Indian Human immunodeficiency virus type 2 (HIV-2) isolate derived lentiviral vector (LV) system reported earlier, we have derived multiple differently configured transfer vectors. Among the features imparted, the novel ones include a blue/white colony screening platform, a shorter vector backbone candidate and availability of default dual tags. Simultaneously, panels with different utilities were also made using this LV. These include neomycin or puromycin or hygromycin selection markers, with options of default promoter, dual multiple cloning site (MCS) availability and drug inducible transgene expression. All the transfer vectors contain the main MCS with the option of single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from cohesive and blunt end cloning sites, as described for the original parent vector. Each transfer vector format was tested by appropriate transgene expression function by transduction of target cells. This is the most comprehensive HIV-2 based lentiviral vector system developed so far and it will significantly aid in preferential applications and thus increase its utility as a versatile system for gene transfer technology.

A lentiviral vector with novel multiple cloning sites: stable transgene expression in vitro and in vivo.

Gene delivery has become an important tool for biological research and gene therapy trials. Lentiviral vector (LV) mediated gene transfer is a preferred approach for stable, sustained transgenic expression. We report here step-wise development of an Indian human immunodeficiency virus type 2 (HIV-2) isolate derived third generation lentiviral vector with a novel, versatile multiple cloning site (MCS) that can also facilitate single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from useful cohesive/blunt end cloning. Efficiency of the vector systems was functionally demonstrated by development of a transgenic enhanced green fluorescence protein (GFP) expressing cell line. Further, a GFP down regulated cell line was derived from the said cell line through LV mediated shRNA expression by cloning the GFP-shRNA cassette using the T/A cloning strategy. Subsequently long term expression of GFP transgene in nude mouse spleen/liver was also documented till 30 days. This LV platform with the enhanced user friendly cloning options will be an important advancement in gene transfer technology.