How to infect 7.5kb insert? - (Nov/06/2009 )
I am working on a large protein whose cDNA size is 7.5kb. And I want to make stable cell lines overexpressing this large protein. Does any know some retrovirus vectors or lentivirus vector can get high infection efficiency with 7.5lb insert?
thanks a lot
Gongfu Panda on Nov 6 2009, 02:58 PM said:
thanks a lot
No one knows?
Clone it into a selectable plasmid (e.g. pBABE) and transfect, it should work. I don't think you need a viral vector.
bob1 on Nov 9 2009, 07:01 PM said:
I want infecet MEF and make stable pools. The transfection efficiency of MEF is relatively low. So I think virus infection would be better.
Most lentiviral vectors have about a 10kb capacity, so your insert should fit without too much trouble.
gfischer on Nov 11 2009, 03:06 PM said:
Thank you for your answer. But I want to use retrovirus instead of Lentivirus. I am wondering: do retorvirus also have 10Kb capacity for insert? I read the detailed information about pLHCX, however, I could not get information about insert size.
Isn't lentivrus a modifed HIV i.e. a retrovirus?
bob1 on Nov 12 2009, 05:12 PM said:
Yes, lentivirus (genome 10kb) is a subfamily of retrovirus. MLV retrovirus have a 8.3kb genome. So lentivirus vector can have a bigger insert than MLV based vectors. But 4.5kb lentivirus vector plus 7.5kb insert exceed the lentivirus genome site, which is my concern. Does anyone have experience to infect large insert such as 7.5kb?