Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)

Genetic switches that can turn on expression of genes of interest in specific tissues or time frames have been used in dissecting biological processes. For example, the yeast-derived transcription factor Gal4 and its upstream activating sequence (UAS) are widely adopted in Drosophila for directing gene expressions.

RMCE was designed to replace existing gene control cassette such as UAS-Gal4 with others through the functions of recombinases, such as Cre, Flp, ?C31. Multiple recombination systems were recently combined to form a flexible enhancer trap, InSITE, for exchanging Gal4 with any other sequence (1). The purpose is to use different existing fly lines that express recombinases in various types of cells.

Related to this topic, UAS-Gal4 was combined with “intra-cassette” recombination for selecting one of the three fluorescent proteins (FPs) in tandem in order to label individual neurons in a setup called “Brainbow”, which was first created in mouse, and recently in flies (2). We expect that InSITE and Drosophila Brainbow would be put together for even broader use of different fly lines and in more cell lineage studies.

One technical barrier for effective use of these systems is the lack of high quality FPs that could provide better brightness and narrower emission spectrum than those currently available. For this reason, the above referenced fly Brainbow research relied heavily on using antibodies against tags fused to the FPs, rendering live cell imaging unobtainable in most cases.

A number of the new FPs in Allele Biotech’s pipeline will be introduced to the field soon, such as the Lancelet YFP, that is ~10X brighter than EGFP, with a very narrow emission bandwidth.

(1) Gahl et Al. 2011, http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1561.html
(2) Hampel et al. 2011, http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1566.html

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Wednesday, March 2nd, 2011 Fluorescent proteins No Comments