| Lentiviral mediated gene transfer has been used recently for gene transfer in lot of mammalian cell line. The most important advantage of using lentiviral vector is their high virus titre and their ability to infect non --dividing cell line. We used this approach to make sh-RNA knockouts of a few signaling proteins from the NFkappaB family. shRNA is used an important genetic tool to silence gene expression in cells to study the phenotype of a particular gene in disease progression and to study potential therapeutic targets for the disease. We made eight copies of the shRNA from the oligonucleotide primers and inserted them into cloning vector which had H1/TO promoter for doxycycline. The expression of sh-RNA was inducible since the promoter was switched on only in the presence of doxycycline. We then cloned this entire shRNA cassette into lentiviral expression (PSLIKneo).This expression vector had antibiotic resistance gene neomycin .Hence cells which had sh-RNA cassette which got successfully cloned into expression vector got selected with neomycin (G418) in this case. We targeted sh-RNA against many NFkappaB signaling proteins like TRAF2, cREL, cIAP1 and cIAP2,IKK1,IKK2,Nemo and caspase 8.We successfully got knockout of cRel and TRAF2 both at the cell survival and protein expression level. We tested whether these genes are involved in the survival of PEL cells. Knocking out these genes showed cell death in the PEL cells. Hence , demonstrating that the expression of these genes are required for the survival of PEL cells. |
