Tag Archives: Viruses

Transcriptional amplification with two rAAVs using tetracycline-controlled gene induction

Cell-type specific expression of genetically encoded indicators or optogenetic probes is often hampered by the use of promoters that are specific but drive expression only weakly. In a recent paper of the Wang Lab at Princeton University, the authors describe a means of introducing a calcium indicator using the TET promoter system in the cerebellar cortex to boost expression by about ten-fold, as determined by quantitative determination of intracellar concentration. Kuhn et al. show specific labeling of Purkinje cells and all interneuron types. Together with a previous paper of the Wang Lab where glial cells were targeted, now nearly all cell types of the cerebellar cortex can be selectively labeled. Additionally, Kuhn et al. overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by systemic pre-injection of hyperosmotic D-mannitol, doubling the time window for functional imaging.

Original article by Kuhn et al.

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Novel AAVs for Cre-dependent control of transgene expression

Recombinant adeno-associated viruses (rAAVs) designed to activate transgene expression in only those cells expressing Cre-recombinase (Cre-On) are widely used to introduce optogenetics constructs into specific cell types and brain regions. In many experiments, what functionally distinguishes Cre-expressing cells from their non-Cre expressing neighbors is not fully understood. A recent paper by Saunders et al. (Sabatini lab, Harvard Medical School) published in Frontiers in Neural Circuits describes two rAAV strategies that allow for simultaneous Cre-On and Cre-Off transgene expression. One strategy (Cre-Switch) achieves differential transgene expression with a single rAAV. The second strategy introduces a Cre-Off vector (FAS), built with lox sites that do not efficiently recombine with loxP or lox2272 sites, which allow FAS rAAVs to be used simultaneously with popular Cre-On DIO (double-floxed inverted ORF) a.k.a FLEX (flip-excision) rAAVs. All Cre-On, Cre-Off, and Cre-Switch rAAV vectors in Saunders et al. are freely available from Addgene.

Original article by Saunders et al.

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