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

Follow AlleleBlog on science and technology: http://allelebiotech.com/blogs/2011/03/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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How to Generate Conditional Knockout Mice with Cre

From AlleleBlog: http://allelebiotech.com/blogs/2010/03/how-to-generate-conditional-knockout-mice-with-cre/

The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.

The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.

The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.

Adenovirus could post a toxicity problem when used at the very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.

To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.

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