Retroviral vector cloning help - (Mar/09/2008 )

I realize this is quite basic, but this is my first cloning with retroviruses. Basically, I am unsure as to how to verify that my gene has been inserted properly into the backbone. I know that the insert is in the proper orientation relative to the 5' and 3' LTRs. However, does it need to be in frame relative to something upstream?

Below are all the details I thought might be helpful at this point.

I have read much of the literature at the Clontech and Nolan group pages, so I am familiar with the principles and important points of retroviral vector expression.

I received the PK1 vector from a collaborator. The vector is MSCV based and is identical to MigR1 except the GFP cassette has been swapped for a Puro cassette.

The MCS of the vector is BglII, XhoI, HpaI, NotI, EcoRI. The EcoRI site is before the IRES and then the Puro. I have cloned into the XhoI site and EcoRI sites. I included a Kozak seq with my gene. The sequence from the BglII reads: agatctctcgagaccATGggg

Many thanks for any help!!

You could do a restriction digest of the retroviral plasmid to confirm presence of the gene, or if needed sequence it. But since you know the orientation of the gene, then I guess the gene is there. Thats what is important. It doesn't have to in frame with anything upstream, but it has to be downstream from the promoter.

Thanks, I guess I should have made it more clear. I have sequenced the insert and that is how I know the orientation and sequence relative to the restriction sites.

I am asking because I am having a hard time seeing any expression when I transfect

I did not see anything in the 293t or Phenix cells after transfection. I have used both fugene and Transit-293 for transfection.

I chose this vector because our collaborator has used it successfully to do virtually the exact same expression. I am just trying to express some point mutants, whereas he expressed the WT allele.

Thanks alot for your suggestions.

How are you detecting your expression? Maybe there's a problem in another part of the procedure?

I detect expression by looking for eYFP expression on our fluorescent microscope. The eYFP is fused to the end of my gene of interest. I have sequenced the insert and eYFP. There are not any stops and everything is in frame. I have used the same sequence with success for transient expression 293 cells.

A western may be a better way to detect.

I agree with the suggestions and will repeat transfection with both the empty vector to make sure that I am generating virus and with a GFP only plasmid to assess transfection efficiency.

Thanks for all your suggestions guys. It is good to have others making me think carefully about the experiment. Best to all.