you have two solution; do a simultaneous co-infection (and mix 1/1) or do a first one, select clones, then do the second transduction... second will take more time but will give you a control (with cells expressing only one receptor) for your future experiments. But the min problem is the selection; whether your vector have different resistance genes (puro, neo, hygro, zeo, blast...) or fluorescnece (gfp and red fp) and you can select with two antibiotics (fluorescence) whether they have the same resistance gene and you will not be sure that the select clone received both viruses (in theory only 1/4 will be double tranduced...) . do you have an easy whay to detect each receptors to be abble to screen clones?

Didier Poncet Thanks for your response!

I have ensured that our markers (truncated receptors) are different for each vector to make the sorting process easier - we currently use a MACS based approach for this.

Have you got any experience in either approach? Would doing the simultaneous co infection drastically reduce my transduction efficiencies of both vectors? I already have a frozen down stock of single transduced cells, could I take those and transduce with the second vector and still get a decent transduction?

If you are using pseudotyped retrovirus (with VSV env protein?) then the transduced cells do not express the env protein so there is no interference so you can do the transduction successively

both our virus is pseudotyped with GaLV.