2 replies to this topic

[Wouter]

Hi all,

I am new to this forum and I hope you can help me out.
I am currently planning to create a pRRLsin cPPT PGK lentiviral construct with an pIRES based insert containing my gene of interest and eGFP.

I will use PCR amplification to amplify my inserts from start till stop codon introducing an Kozak sequence before the atg and the desired restriction sites.
Subsequently I will use restriction enzymes to place the complete insert: kozak"gene of interest"-IRES-kozak"eGFP" into the pRRLsin.

My question, concers the IVS synthetic intron that is present in the pIRES plasmid from clontech. Am I required to also introduce this IVS sequence into the pRRLsin? According to the manual of the pIRES it is there to enhance RNA stability. I however have not found a restriction site that allows direct cloning from the pIRES to the pRRLsin including this IVS sequence.

I do however have a site (introduced through my primer) that allows direct cloning of the insert whithout the IVS sequence.
The complete insert is too long to amplify through PCR approximately 2.5kb.

Does anyone have axperience with this IVS sequence and its effect on protein expression. If I don't really need it, it would save me a lot of cloning. All suggestions would be very much appreciated!!

Thanks

Wouter

[NemomeN]

To make a long winded explanation short, you don't need it for what you are doing.

[Wouter]

That's exactly what I wanted to hear!
Thank you very much for your quick reply.

Wouter