Poor expression in my cloned vector (pMSCV-PIG) - (Mar/05/2013 )

Dear experts,

I have cloned my gene of interest into pMSCV-PIG vector. The brief insert sequence is as followed;

CTCGAG(= XhoI) GCCACC (=Kozak) ATG-------TAA (ORF) GTTAAC (=HpaI)

I used two enzyme sites for this cloning (XhoI and HpaI). In transient transfection in 293T cells, I could not get any overexpression.

I haven't used pMSCV-PIG vector before, so don't know if inserting ORF without extra mRNA sequence at 5' and 3' ends is enough.

Could anyone tell me any suggestion?

Thanks.

Cancerous

It should be fine to insert without any UTRs.

What times have you tried to detect the protein at following transfection? Have you looked to see if the mRNA is expressed?