need some help in this reading frame - (Mar/21/2006 )
Hi. I need badly some help.
I cloned cDNA of 2 hormones into retroviral vectors (pBABE puro) which have LTR promoters (very active theoretically
nevertheless, I was not able to find any overexpression and am in doubts if its the reading frame which went wrong.
I cloned in to the EcoRI sites of the vector and I was told that with my starting codon atg (which is 253 bp away from the ecoRI cutting site) will be enough to initiate translation in the correct frame.
is this true?
do we have to consider the reading frame in this case?
thanks
The reading frame has little to do with your problem: you are not altering anything in the coding region or putting a tag on it, so the reading frame is not the first thing to worry about.
You mention that you can check for expression. I supose you mean for the hormone with eg an antibody: Wonder if you first made virus, transduced the target cell, selected them w/ puro and then tested the expression of your homone. You see, there are many steps which could go wrong before you do your test.
So maybe it is best to do something like this: transfect eg 293T cells w/ your construct together with a GFP expressing construct (1:1). Wait 48h and check for GFP expression under a fluorescent microscope (~50% of cells should be positive). Then test the cells for the expression of your hormone. You should get a major signal. If there is none then there is something wrong with the cloning of the cDNA into pBabe. If you do get a good signal the problem lies w/ a later step.
Some features hat should be avoided or are incompatible in/with retroviral vectors are things like (cryptic)splice donors/acceptors, repeated sequences and polyA signals. All complete cDNA's do have a poly-A signal ATAA (or something) about 30 bp from the polyA tail. The cDNA is missing the GC rich region after the polyA signal, but it will often reduce virus production. Some poly-A signals do not seem to matter very much (people make cDNA libraries in retrovirusses....) while other (strong) polyA signals prevent any virus to be produced (so no read through, through the poly-A signal). So you can check for such features in your cDNA and remove them.
Good luck
Thanks a lot ilopostino, Ill try the gfp transductions
You mention that you can check for expression. I supose you mean for the hormone with eg an antibody: Wonder if you first made virus, transduced the target cell, selected them w/ puro and then tested the expression of your homone. You see, there are many steps which could go wrong before you do your test.
So maybe it is best to do something like this: transfect eg 293T cells w/ your construct together with a GFP expressing construct (1:1). Wait 48h and check for GFP expression under a fluorescent microscope (~50% of cells should be positive). Then test the cells for the expression of your hormone. You should get a major signal. If there is none then there is something wrong with the cloning of the cDNA into pBabe. If you do get a good signal the problem lies w/ a later step.
Some features hat should be avoided or are incompatible in/with retroviral vectors are things like (cryptic)splice donors/acceptors, repeated sequences and polyA signals. All complete cDNA's do have a poly-A signal ATAA (or something) about 30 bp from the polyA tail. The cDNA is missing the GC rich region after the polyA signal, but it will often reduce virus production. Some poly-A signals do not seem to matter very much (people make cDNA libraries in retrovirusses....) while other (strong) polyA signals prevent any virus to be produced (so no read through, through the poly-A signal). So you can check for such features in your cDNA and remove them.
Good luck
Just to make sure because people use terms differently: I ment a transient test, with a transient cotransfection (mixing a GFP construct with your construct). Not a viral transduction. But i suppose that was clear. Anyway good luck.