You should produce (package) the viruses separately as cotransfection will lower the yield of the individual viruses. You could try to concetrate the virus stocks in a centrifugal filter unit e.g. Amicon ultra with a 100kDa molecular weight cut off prior to use. Another possibility is to use "spinoculation" i.e. low speed centrifugation in a swing out rotor to force the virus onto the cells. In this way a much larger volume can be used. I  regularly spinoculate mammalian cells by a 1 h centrifugation at 2900rpm in a Beckman JS 5.9 rotor.

Best,

Mathilda

Recombination is a very distinct possibility when you have closely related vectors that you are trying to package in a single transfection. Recombination may occur both at the transfection stage and the reverse transcription stage. It will be better if you packaged the vectors individually. As Mathilda has suggested, you could concentrate the virus if you are trying to get your infection to a higher level. Else, you could sort the cells post transduction with the help of markers.

Since you are working with HSC cells, I do not know if spinoculation would affect the 'stemness' of the cells later on. Use of polybrene to enhance the infection may also affect the 'stemness'. You could search the literature for other means of increasing infection efficiency in your HSCs. [I believe RD114 Envelope works best for HSC infections].

Good luck.

Thank you for your helpful answers, colleagues! Now it is obvious for me that packaging should be done separately. I use polybrene, I also tried centrifugation of virus at 20 000 g for 1.5 h, and it greatly enhanced infection rate, so now I do not think that I will expect difficulties with gene delivery.  

in pooled sh / sg RNA screen, 100s to 10000s of different viruses are packaged together, so I think you should be fine doing it together... - that's at least my opinion

Yes, but if you have optimized the amount of plasmid added to the cells (as you should) to get the highest vector production, adding a second plasmid will do no good. The total number of vectors packaged is likely to be the same, only it will now be of two different kinds, i.e. lower amounts of each.

sure, but if you add 2 separately, you will also have lower amounts of each if you end up adding the same valume of virus supernatant / concentrated virus  (in case your cells are rather sensitive) - or you just double up, but that you could also do with the pooled super 

Thank you for your suggestions!

@ Bastian

thanks for the mail - in case ppl also read Denning et al., and wonder which DEAE it is eactly, I just contacted the last author and he told me it's simply DEAE-Dextran hydrochloride (D9885 SIGMA) - would be if ppl would share their experiences with DEAE vs polybrene...

When any retrovirus is assembled in the cytoplasm post replication, the gag molecule will package any RNA (its viral RNA or cellular RNA).  The gag molecule cannot differentiate between cellular and viral RNA.  In that case, I would expect a cell co-infected with two different retroviruses to package mixed RNA into their virions.  Their viability and infectivity along with genetic recombinations just opens a new can of worms by each of themselves.

well, but isn't it just one copy per virion anyways? besides, I stick with what I said - the whole CRISPR and shRNA screening field is partly based on the fact that it seems to work just fine - I know that's not a satisfying explanation, but maybe we can be a little pragmatic here as well

For retroviruses, it is two copies of viral genome. Gag has a nucleo capsid region which binds to the packaging element of the viral genome and this interaction is extremely specific. While, non viral RNA can get into the viral particles, they are in the virions only because of abundance in the cytoplasm and not because retroviruses pack any RNA. Reverse transcriptase of retroviruses are remarkably good at recombination. Hence if you have two different genomes in a single virus, with sufficient overlap, you will get recombination during reverse transcription.