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The iCreERT2-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 (Shimshek et al. 2002) is fused with the ligand-binding domain of the estrogen receptor (ER) to obtain a ligand-inducible recombinase. 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 iCreERT2-FRT-neo-FRT template encodes the neomycin/kanamycin resistance gene (aminoglycoside phosphotransferase) 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 template can easily be used to generate targeting constructs mediated by a single Red/ET Recombination step. The iCreERT2-FRT-neo-FRT template is not linear but plasmid based (4185 bp in size). Due to its R6K origin it can¡¯t replicate in most of the frequently used 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 1µl per reaction as template.

iCreERT2-FRT-neo-FRT

 

 

 

 

 

 

ATGGTCTCCAACCTGCTGACTGTGCACCAAAACCTGCCTGCCCTCCCTGTGGATG CCACCTCTGATGAAGTCAGGAAGAACCTGATGGACATGTTCAGGGACAGGCAGGC CTTCTCTGAACACACCTGGAAGATGCTCCTGTCTGTGTGCAGATCCTGGGCTGCC TGGTGCAAGCTGAACAACAGGAAATGGTTCCCTGCTGAACCTGAGGATGTGAGGG ACTACCTCCTGTACCTGCAAGCCAGAGGCCTGGCTGTGAAGACCATCCAACAGCA CCTGGGCCAGCTCAACATGCTGCACAGGAGATCTGGCCTGCCTCGCCCTTCTGAC TCCAATGCTGTGTCCCTGGTGATGAGGAGAATCAGAAAGGAGAATGTGGATGCTG GGGAGAGAGCCAAGCAGGCCCTGGCCTTTGAACGCACTGACTTTGACCAAGTCAG ATCCCTGATGGAGAACTCTGACAGATGCCAGGACATCAGGAACCTGGCCTTCCTG GGCATTGCCTACAACACCCTGCTGCGCATTGCCGAAATTGCCAGAATCAGAGTGA AGGACATCTCCCGCACCGATGGTGGGAGAATGCTGATCCACATTGGCAGGACCAA GACCCTGGTGTCCACAGCTGGTGTGGAGAAGGCCCTGTCCCTGGGGGTTACCAAG CTGGTGGAGAGATGGATCTCTGTGTCTGGTGTGGCTGATGACCCCAACAACTACC TGTTCTGCCGGGTCAGAAAGAATGGTGTGGCTGCCCCTTCTGCCACCTCCCAACT GTCCACCCGGGCCCTGGAAGGGATCTTTGAGGCCACCCACCGCCTGATCTATGGT GCCAAGGATGACTCTGGGCAGAGATACCTGGCCTGGTCTGGCCACTCTGCCAGAG TGGGTGCTGCCAGGGACATGGCCAGGGCTGGTGTGTCCATCCCTGAAATCATGCA GGCTGGTGGCTGGACCAATGTGAACATTGTGATGAACTACATCAGAAACCTGGAC TCTGAGACTGGGGCCATGGTGAGGCTGCTCGAGGATGGGGACCTCGAGCCATCTG CTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTC TAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCTTG TTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCA GTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCA CATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGAT CAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCT GGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGA CAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTG GCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCC TCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCT GAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGAC ACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGC GGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGG CATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTG CTGCTGGAGGCGGCGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGG CATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATC GCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACA GCTTGATGAAGATCTGAGCTCCCTGGCGGAATTCGGATCCAGATCTTATTAAAGC AGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTC ATCAATGTATCTTATCATGTCTGGTCGATAATACGACTCACTATAGGGCTCGAGG AAGTTCCTATACTTTCTAGAGAATAGGAACTTCCGCGCCGCACACAAAAACCAAC ACACAGATCATGAAAATAAAGCTCTTTTATTGGTACCGAATTCGCCAGGGAGCTC TCAGACGTCGCTTGGTCGGTCTTTATTCGAACCCCAGAGTCCCGCTCAGAAGAAC TCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGT AAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACG GGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCG ATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGC CATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAA CAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACA AGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGT CGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCAT GATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGC ACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAG CTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTG CAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCC TGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATCGTCTGTTGTGCCC AGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCC ATCTTGTTCAATGGCCGATCCCATGGTTTAGTTCCTCACCTTGTCGTATTATACT ATGCCGATATACTATGCCGATGATTAATTGTCAACACGTGCTGCTGCAGGTCGAA AGGCCCGGAGATGAGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCG TGCAGAATGCCGGGCCTCCGGAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCC GCCCCTGAGCCCGCCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCG AAGGAGCAAAGCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCT CAGCGGTGCTGTCCATCTGCACGAGACTAGTGAGACGTGCTACTTCCATTTGTCA CGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGGGAGGA GTGGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGG TGGATGTGGAATGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAG CGGGGCTGCTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACC CGGTAGAATGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGCGGCGCCCTTT

AGTGAGGGTTAATT