Variable recombination efficiency in responder transgenes activated by Cre recombinase in the vasculature.
Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene expression in a tissue-specific manner. We report analysis of two SM22alphaCre lines and their ability to mediate genomic recombination in five independent Cre-responsive transgenic lines. One of the SM22alphaCre lines developed was a tet-on system based on the reverse tetracycline transactivator. Our goal was to use this strategy to inhibit the Notch signaling pathway specifically in smooth muscle cells. Our responder transgenes contained a constitutively expressed marker gene (chloramphenicol acetyltransferase, CAT), flanked by loxP sites in direct orientation, upstream of Notch-related transgenes. We developed two dominant negative Notch transgenic responder lines activated by Cre-mediated DNA recombination. The first is the extracellular domain of human Jagged1, and the second is the extracellular domain of the human Notch2 receptor. Despite high expression of the marker gene in all responder lines, we found that Cre-mediated genomic recombination between these five lines was highly variable, ranging from 46 to 93% of individuals using an SM22alphaCre activating strain, or 8-58% of individuals using an inducible SM22alphartTACre. In all cases examined, detection of recombination by PCR correlated with expression of the transgene as determined by Western blot analysis. Our studies reflect the variability in recombination success based on the responder strain, presumably due to inaccessibility of the locus of integration of the responder allele.
Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA.
Bochiwe
B
Hara-Kaonga
Yu Alice
YA
Gao
Matthew
M
Havrda
Anne
A
Harrington
Ivy
I
Bergquist
Lucy
L
Liaw
D000818:Animals; D002460:Cell Line; D005260:Female; D017930:Genes, Reporter; D019426:Integrases; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D032389:Myocytes, Smooth Muscle / Q000201:enzymology / Q000378:metabolism*; D051880:Receptors, Notch / Q000037:antagonists & inhibitors; D011995:Recombination, Genetic; D015398:Signal Transduction / Q000502:physiology; D019076:Transgenes
D051880:Receptors, Notch; C045073:Cre recombinase; D019426:Integrases