Hello all,

I would like to get some clarity on a fundamental biological process of lenti/retroviruses and stable-cell line generation using 3rd generation system .

1. During the production of viral particles from HEK293T/FT, using the 3rd generation lentivector system (to generate stable cell lines), what happens if there are 5 copies of AUAAA introduced in the middle of the viral genome in the transfer plasmid? Can successful and infectious virion particles be made using such a construct and later can an integrated stable cell line be possible to make?

2. Is it possible that the same cell (HEK293T/FT) which produces the lentiviral particles can also get re-infected by the virus (if the viral sup is not removed for extended time) and the RNA genome gets reverse transcribed and integrated in the producer cells itself?

3. Is it possible that if the vector in point no. 1, which also has a puromycin resistance marker be transfected into HEK293T/FT and passaged for several generations with puromycin could generate a stable cell-line in those cells?

Would appreciate any input along with appropriate reference.

Thanks