This is probably a daft question; I have cloned in a gene of interest to a lentiviral expression vector and successfully made lentiviral particles that easily transduce HEK cells producing the expected protein (confirmed on WB). However I cloned in my gene of interest as a DNA sequence, and am now wondering the mechanism of viral integration in to the genome? Did the DNA simply get introduced without any involvement of the reverse transcriptase steps or did the cell simply ignore that the gene of interest was DNA not RNA and go through the rev transcriptase steps before integrating it in to the host cell genome?
Hi there,
When you cloned your gene of interest into lentiviral vector, you cloned it into a plasmid (DNA). Then the plasmid is transfected into a packaging cells, where the viral genome (including your gene of interest) is transcribed from DNA into RNA and packaged into functional virus particles. So when you have your virus particles it is all RNA. When the virus infects a host cell the capsid is unpacked and RNA genome is reverse transcribed into DNA, which then gets integrated into nucleus.
I hope that helps
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