—Frequently Asked Questions—

Regulatable Vectors

1. Q: Are the maps that are found on this web site absolutely reliable?
   A: While we made every effort to produce reliable maps, we still find the occasional surprise...Those that we recently found out about and that did not make it into the maps yet are following.
   HRSp: an extra NotI site has been found in the plasmid backbone, next to the AMP resistance, on the opposite side as the 3' LTR
2. Q: How do you package the viruses?
   A: We routinely use the Phoenix packaging cell line, which can be obtained from Garry Nolan's lab at Stanford University (http://www.stanford.edu/group/nolan/). Also look up the appropriate protocols on the Nolan web site.
3. Q: Is there a selectable gene in the RTAb viruses?
   A: No, we never needed to select cells because we typically obtain over 90% infection efficiency using these viruses. This may not hold true for every cell type.
4. Q: Can I change the puro resistance in HRSp with another selectable marker?
   A: Why not?
5. Q: Can I replace the EGFP in HRIgfp with another selectable marker?
   A: Sure.
6. Q: Should the HRIgfp and HRSp-GFP express GFP in transtient transfections?
   A: Yes. Self-inactivating viruses only loose their 5' LTR after reverse transcription, therefore they will not be regulatable in transient transfections.
7. 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
8. 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.
9. 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.
10. Q: Does the system work in vivo?
    A: The MLV LTRs used to drive expression of the transactivator is much less strong in vivo than in vitro. Furthermore its activity may vary with the type and the differentiation state of the cells. In summary, it is quite possible that even cells that display exquisite regulation in vitro will misbehave once transplanted in vivo.