Cre recombinase activity is inhibited in vivo but not ex vivo by a mutation in the asymmetric spacer region of the distal loxP site.
The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region "ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3') loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl-tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue-specific promoters: calcium/calmodulin-dependent kinase II alpha (Camk2a-cre) and myosin light polypeptide 1 (Myl1-cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt((loxP/loxP)) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695-700, 2015. © 2015 Wiley Periodicals, Inc.
Department of Human Genetics, Human Genetics and Genomics Graduate Program, University of Miami, Miller School of Medicine, Miami, Florida.
Tania
T
Arguello
Department of Neurology, University of Miami Miller School of Medicine, Miami, Florida.
Carlos T
CT
Moraes
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D021901:DNA, Intergenic / Q000378:metabolism*; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics; D019426:Integrases / Q000378:metabolism*; D018345:Mice, Knockout; D008822:Mice, Transgenic; D017354:Point Mutation
D021901:DNA, Intergenic; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase; C045073:Cre recombinase; D019426:Integrases
cre-lox; mitochondria; recombination