Should the HRIgfp and HRSp-GFP express GFP in transtient transfections?
Yes. Self-inactivating viruses only loose their 5′ LTR after reverse transcription, therefore they will not be regulatable in transient transfections. • Q: How should we expand the plasmids? A: All the plasmids are high copy number except the RTAb plasmids…. from those, expect a very low yield. The details: Strain: usually, we use recombinations deficient strains (such as Stbl-2 from Gibco or Sure from Stratagene) although we never had recombination problems with this plasmids and a normal bacterial strain may be just fine. Medium: (Yes, I get asked this too) normal LB. Resistance: AMP • Q: What primers should we use to sequence from HRSp series plasmids? A: While these may not be the best primers (they are the first and only we tested) they work well enough. • Q: Is it OK to move the transactivator in another vector? A: The expression level of the transactivator is critical: you can put it in any vector, but make sure is under a strong promoter. • Q: Does the system work in vivo? A:
