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.
Santhosh
C V
CV
Virology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra 410 210, India.
Tamhane
Mayur C
MC
Kamat
Rohan H
RH
Patel
Vainav V
VV
Mukhopadhyaya
Robin
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015498:HIV-2; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000235:genetics; D008969:Molecular Sequence Data; D019076:Transgenes
D035683:MicroRNAs; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins