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Mechanism of Viral Transduction

HEK293FT transduction

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#1 Benny85

Benny85

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Posted 14 March 2012 - 09:45 PM

I'm using 3rd generation lentivirus plasmids (packaging plasmid 12251, 1253, 12259 - from Addgene) to transfect HEK293FT cells to make lentiviruses.

12251: CMV-GAG-POL
12253: CMV-Rev
12259: CMV-VSVG
Transfer plasmid

What I understand is that during the assembly of lentivirus in the packaging cell line HEK293FT, only the Transfer plasmid is packaged as a RNA form into the virus (because lentivirus is RNA virus, so RNA is packaged). No packaging plasmids is packaged. So when the RNA is being introduced into the host cell ( I'm using human BJ fibroblasts), how is it going to be reverse transcribed? And how is the reverse transcribed RNA (cDNA) going to be integrated into the host genome when integrase is not present? Because reverse transcriptase and integrase genes are not packaged into the virus. I know that the Transfer plasmid construct is flanked by LTR repeats and integration sequences, does it mean that the transgene is integrated into host genome randomly by random recombination? is there even such thing as random recombination? How does this occur (integration sequence in the transfer plasmid line up with the recombination sequence on the fibroblast genome and recombination occur)?

Another question, the transgene is cloned in the viral vector/plasmid. When packaging, does the whole RNA form of the viral vector and transgene construct get clones into the newly formed virus or only the transgene construct?

I'm very puzzled. Please help me. thanks.





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