The technology of gene inactivation (knockout, KO) has boosted our understanding of the role of a number of genes in vivo. However, following gene deletion, the interpretation of the results may be complicated by the existence of compensatory adaptation at different network levels (i.e., nuclear gene networks, organ-to-organ endocrine network, central nervous synaptic networks and so on). A classic approach to limit some of those problems is to choose a conditional KO strategy in desired tissues or at desired time-points, in order to observe the phenotypic pictures before development of functional adaptations in response to gene inactivation.
The site-specific Cre-loxP system is widely used for various methods of genome engineering, and lot of transgenic lines express Cre recombinase in a tissue-specific manner. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Dated 1999, the most known test line is the Soriano's R26R (Rosa26 lacZ) in which beta-galactosidase is expressed following Cre recombination.
|A green central nervous system marked by GFP after|
Nestin-Cre-recombinase action on IRG developing mouse
Recently, a new reporter line has been described: the IRG mouse. Compared to R26 reporter, IRG adopt insulators instead of locus strategy (see the previous post where do you express your transgene?). In this line, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and shifted to an enhanced green fluorescent protein (EGFP) following recombination. For instance in the picture, you can see Cre-mediated recombination in CNS (nestin positive cells) of an embryo at day 13.5 of development. Given the distinct advantages of fluorescent reporters over the lacZ staining (no need for exogenous substrates, fluorescence can be visualized in tissues without fixation artifacts and cells can be isolated using FACS sorting), the IRG mouse will bring soon new adepts to the emerging field of optical imaging.
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- Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genetics, 21(1), 70-71. DOI: 10.1038/5007
- De Gasperi, R.,[...] Elder, G.A. (2008). The IRG mouse: A two-color fluorescent reporter for assessing Cre-mediated recombination and imaging complex cellular relationships in situ. Genesis, 46, 308-317. DOI: 10.1002/dvg.20400