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How to infect 7.5kb insert?


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#1 Gongfu Panda

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Posted 06 November 2009 - 12:58 PM

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

#2 Gongfu Panda

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Posted 09 November 2009 - 11:48 AM

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



No one knows?

#3 bob1

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Posted 09 November 2009 - 05:01 PM

Clone it into a selectable plasmid (e.g. pBABE) and transfect, it should work. I don't think you need a viral vector.

#4 Gongfu Panda

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Posted 11 November 2009 - 12:10 PM

Clone it into a selectable plasmid (e.g. pBABE) and transfect, it should work. I don't think you need a viral vector.


I want infecet MEF and make stable pools. The transfection efficiency of MEF is relatively low. So I think virus infection would be better.

#5 gfischer

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Posted 11 November 2009 - 01:06 PM

Most lentiviral vectors have about a 10kb capacity, so your insert should fit without too much trouble.
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#6 Gongfu Panda

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Posted 11 November 2009 - 02:34 PM

Most lentiviral vectors have about a 10kb capacity, so your insert should fit without too much trouble.


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.

#7 bob1

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Posted 12 November 2009 - 03:12 PM

Isn't lentivrus a modifed HIV i.e. a retrovirus?

#8 Gongfu Panda

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Posted 13 November 2009 - 07:54 AM

Isn't lentivrus a modifed HIV i.e. a retrovirus?



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?




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