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.

Full-length genome characterization of an HIV type 2 isolate from India.

An infectious Indian human immunodeficiency virus-2 (HIV-2) subtype A isolate was completely sequenced and analyzed and its phylogenetic relatedness was investigated. The unusual limited size of the long terminal repeat (LTR) from the isolate was caused due to a truncation within the nef open reading frame (ORF) located at the U3 region of the LTR. The genetic relatedness and lineage of this HIV-2 strain were investigated. The close relatedness of this isolate to West African HIV-2 isolates confirms a geographical entry route of HIV-2 to this part of the Indian subcontinent. This is the first report of an HIV-2 full genome analysis from the Indian subcontinent as well as from Asia.