iCre-FRT-neo-FRT PCR template is designed to facilitate the insertion of such a functional cassette into targeting constructs by Red/ET recombination.
A codon-improved version of P1 bacteriophage derived Cre-recombinase is located upstream of the neomycin/kanamycin resistance gene (aminoglycoside phosphotransferase). Mammalian codon usage was applied for the altered Cre version (iCre). By introducing silent base mutations the high CpG content of the prokaryotic coding sequence was reduced, thereby reducing the chances of epigenetic silencing in mammals (Cohen-Tannoudji et al., 2000).
The iCre-FRT-neo-FRT template encodes the neomycin/kanamycin resistance gene which combines a prokaryotic promoter (gb2) for kanamycin resistance in E.coli with a eukaryotic promoter (PGK) for neomycin resistance in mammalian cells.
The prokaryotic promoter gb2 is a slightly modified version of the Em7 promoter; it mediates higher transcription efficiency than the normally used Tn5 promoter. The promoter of the mouse phosphoglycerate kinase gene (PGK) is used as eukaryotic promoter. A synthetic polyadenylation signal terminates the kanamycin/neomycin transcription. The cassette is flanked by FRT sites for later excision by Flp-recombinase.
Using the provided PCR template one can easily create an iCre-FRT-neo-FRT cassette flanked by homology arms to insert the cassette by Red/ET recombination into the vector of choice.
The iCre-FRT-neo-FRT template is not linear but plasmid based (3446bp in size). Due to its R6K origin it can't replicate in most of the common E. coli strains. The PCR product can therefore be used directly for downstream applications without any further purification. At least 20 PCR reactions can be performed using 1ul per reaction as template.