I'm planning to do a lentiviral transfection of mouse bone marrow progenitors for in vivo studies in reconstituted mice. Our lab has this working for expression of shRNA, but we haven't attempted to express large products. We ultimately want to use the system to simultaneously express 2 fluorescent protein tagged proteins, each ~4kb (including the fluorescent protein), to do imaging studies.
The issues/questions I have:
1. If both proteins are included in one lentiviral construct, it will be large and so transfection efficiency may be low. Would using 2 separate vectors (1 for each protein) make more sense? We can deal with low transfection by enriching transfected cells before injecting back into the mouse for hematopoietic reconstitution.
2. What's the best way to achieve relatively equal expression of both proteins? The standard bicistronic vectors with IRES have much lower expression of the protein following the IRES (which doesn't really matter when it's just a reporter, but does matter in my case). Is there a lentiviral vector with dual promoters? What about using a mix of lentiviral vectors with gene A after promoter and gene B after IRES in one vector and the opposite order in the other vector?
Thanks!
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