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Retroviral Vector Construction


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8 replies to this topic

#1 tinker

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Posted 26 July 2010 - 02:45 PM

Is it possible to express an ORF and an sh-RNA sequence from the same vector? I know there are plasmids with different integration sites for multiple shRNA sequences but can I use the same vector to express one gene while knocking down another?
Could it be as simple as dumping in my gene and promoter from one retroviral vector into my pSuper plasmid or is it way more difficult?

Thanks.

#2 laurequillo

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Posted 27 July 2010 - 02:41 AM

Maybe you can use the plasmid you use for silencing and remove the puro selection for the protein you want to express? I dont really know, but I am looking foward to reading what people think!
I am not sure about the use of bicistronic vectors for what you want to do...
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#3 Rsm

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Posted 27 July 2010 - 09:18 AM

Yes, you can use both from one vector. You'll have to use different promoters, though (correct me if I'm wrong). PolIII promoter for siRNA and PolII promoter for ORF. You can then knock down your gene of interest and overexpress, for example, a kinase-dead mutant of your gene of interest. You'll need to target the UTR obviously... Haven't done it yet, but know some people who had some success with it.

Cheers,

Minna
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#4 Rsm

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Posted 27 July 2010 - 09:27 AM

Right, here you are: http://www.addgene.o...dentifier=11643
Even better, Lentiviral vector with a TetON shRNA and ORF of your choice.
The cloning was a bit difficult, if I remember correctly.
pLVCT-tTR-KRAB from http://tronolab.epfl.ch/

Cheers,

Minna
I got soul, but I'm not a soldier

#5 laurequillo

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Posted 27 July 2010 - 10:48 AM

Yes, you can use both from one vector. You'll have to use different promoters, though (correct me if I'm wrong). PolIII promoter for siRNA and PolII promoter for ORF. You can then knock down your gene of interest and overexpress, for example, a kinase-dead mutant of your gene of interest. You'll need to target the UTR obviously... Haven't done it yet, but know some people who had some success with it.

Cheers,

Minna


Yeah you will need pol II and III promoters. It is cool to know that there is such vector available! :D
"He must be very ignorant for he answers every question he is asked" Voltaire

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#6 tinker

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Posted 27 July 2010 - 11:25 AM

Right, here you are: http://www.addgene.o...dentifier=11643
Even better, Lentiviral vector with a TetON shRNA and ORF of your choice.
The cloning was a bit difficult, if I remember correctly.
pLVCT-tTR-KRAB from http://tronolab.epfl.ch/

Cheers,

Minna



Thanks for the information, close to what I'm searching for. It looks like they have a constitutive vector which would be more appealing. One thing I've noticed is the lack mammalian drug selectable marker, unless I'm missing it. They used GFP as their ORF and selectable marker.

#7 Rsm

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Posted 28 July 2010 - 02:02 AM

"Yeah you will need pol II and III promoters. It is cool to know that there is such vector available! :D"

Hmmm, I am just not so sure. Weren't there some PolII promoter that could also transcribe shRNA? I remember that c-Myc is transcribed by both PolII and PolIII. Or so they say.

Edited by Minna, 28 July 2010 - 02:02 AM.

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#8 laurequillo

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Posted 28 July 2010 - 02:28 AM

"Yeah you will need pol II and III promoters. It is cool to know that there is such vector available! :D"

Hmmm, I am just not so sure. Weren't there some PolII promoter that could also transcribe shRNA? I remember that c-Myc is transcribed by both PolII and PolIII. Or so they say.


I will have to check it.
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"

#9 laurequillo

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Posted 28 July 2010 - 02:34 AM

Yes indeed, you have shrna driven by pol ii promoters. I did not know that
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"




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