#Nox brighter screen full#

Less common and technically still challenging applications include the use of Cre to generate chromosomal alterations, such as large deletions or inversions, as well as inter- and intra-chromosomal translocations to mimic human diseases, like for instance leukemias 6.ДЄ very promising application of the Cre/loxP recombination system, which has not yet been fully exploited, is its use as a tool for lineage-tracing and fate-mapping studies 5.

Also, conditional deletion of floxed genomic DNA sequences has become a rather standard approach for assessing tissue-, cell-type- or developmental stage-specific gene functions, provided appropriate Cre-transgenic mouse strains are available. Cre-mediated removal of loxP-flanked (floxed) selection cassettes, which are invariably introduced during the process of gene targeting into the mouse genome, and which may have unpredictable effects on neighboring genes, is already a routine manipulation. In combination with gene targeting techniques, the Cre/loxP recombination system has revolutionized the genetic analysis of mice 4, 5.

Importantly, both activity and specificity of Cre recombinase are retained in eukaryotic cells 3, permitting the use of Cre as a genetic engineering tool in any cellular context. In contrast, DNA flanked by two oppositely oriented sites will be inverted, a fully reversible process. The result of Cre-mediated recombination between two loxP sites depends on their specific orientation relative to one another: a DNA sequence flanked by two directly repeated loxP elements is cut out as a circular molecule, an effectively irreversible reaction due to loss of the excised product. LoxP sites are composed of two 13-bp inverted repeats and an asymmetric 8-bp spacer sequence, which endows individual loxP elements with a defined orientation 2. Taken together, the data show that our C57BL/6-inbred reporter mice are ideally suited for sophisticated lineage-tracing experiments requiring sensitive and quantitative detection/purification of live Cre-expressing cells and their progeny.Д¬re recombinase is a small, bacteriophage P1-derived integrase that catalyses defined DNA recombination events between specific target sites, termed loxP (locus of crossover in P1) 1.

#Nox brighter screen full#

Cytofluorometric and histological analyses illustrate: (i) non-toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre-mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding вЂњleakyвЂќ reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP-expressing cells even in paraformaldehyde-fixed tissue sections, and (v) full compatibility with GFP/YFP-based fluorescent markers in multicolor experiments. We directly demonstrate the usefulness of our reporter strain in inter-crosses with a вЂњuniversal Cre-deleterвЂќ strain and with mice expressing Cre recombinase in a T lineage-specific manner. By targeting a tandem-dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6-ES cells, we have generated a novel inbred Cre-reporter mouse with several unique characteristics. The considerable potential of Cre recombinase as a tool for in vivo fate-mapping studies depends on the availability of reliable reporter mice.