The idea would be to use retroviral vectors bearing LoxP sites to infect cells expressing inducible Cre recombinase (under control of Dox or Tamoxifen). This would be used to delete transgene expression in vivo at a desired time point.
Is this safe or would multiple retroviral integration sites cause major chromosomal deletions and/or rearrangements?
Indu Verma's lab describe a self-deleting Cre lentivirus (PMID: 11553794) way back in 2001 that worked quite well. There is no reason to think that this wouldn't work for other types of retrovirus, and in fact, I wouldn't be surprised if a careful literature search turns up reports of this strategy. While the reference I cite here is different from the experimental design you propose, it is the same proof of principle (multiple integration sites are not excessively problematic).
Indu Verma's lab describe a self-deleting Cre lentivirus (PMID: 11553794) way back in 2001 that worked quite well. There is no reason to think that this wouldn't work for other types of retrovirus, and in fact, I wouldn't be surprised if a careful literature search turns up reports of this strategy. While the reference I cite here is different from the experimental design you propose, it is the same proof of principle (multiple integration sites are not excessively problematic).
Hi,
Lentiviral vectors in combination with Cre-lox system have been used recently in a very interesting paper.
Here goes the link:
http://www.pnas.org/content/110/17/7038.full?sid=ba3b4ec5-aff2-42fc-9968-0ef8f644d899
In our lab we routinely use an MSCV-Cre virus, which has the tendency to get silenced often after some culture or in vivo. Galla et al. (http://www.ncbi.nlm.nih.gov/pubmed/15494317) have developed a RNA mediated transfer system and compared it to a integration deficient retrovirus. Both systems do not have continuous Cre expression, and should therefore be less cytotoxic.
In our case the Cre expression would only be transient to remove the transgene from the integrated retroviral sites. I was just wondering if the different integration sites in a single cell could cause chromosomal mayhem when Cre is around.
We had some trouble removing multiple (up to 4) floxed sites in a virus using Cre. Depending on the chromosomal location, I think, some sites are less accessible than others. In a Cre mediated knockout in a mouse, this seems to be less than an issue. Flp recombinases might also work well with multiple insertions
I agree with Martijn that chromatin context may affect accessibility to Cre. On the other hand, inaccessible integrants may also fail to express your transgenes, so would be functionally inactive, and therefore not much of a concern.
You can partially mitigate these problems by either using site-specific integration or simply using a low MOI for your inducible transgene.
A separate, but important issue, is that the tet-transactivator is not well tolerated by all cell types. And expression of rtTA or tTA-VP16 fusions can alter cell behavior independently of the inducible transgene. Depending on the broader context of your experimental design, this may or may not be an issue. However, before designing a multi-component Cre/lox/tet-inducible-GOI system, you should make sure the tet-transactivator does not interfere with the cell behavior or phenotype you wish to study.
Thank you Daniel.
I am currently using a Tet-ON system in which the rtTA is encoded in the retroviral vector together with the target gene. Amongst several concerns of the current system one of them is exactly what you mentioned: I believe the transduced cells are not behaving normally. Perhaps a tamoxifen induced Cre would be less of an issue.
Again, thank you.
Yes, I think the problems with rtTA-VP16 are under-reported. ER-Cre might be a good solution.
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