Activation kinetics and off-target effects of thymus-initiated cre transgenes.
The bacteriophage enzyme Cre is a site-specific recombinase widely used to delete loxP-flanked DNA sequences in lineage-specific fashion. Several mouse lines that direct Cre expression to lymphoid progenitors in the thymus have been established, but a side-by-side comparison of when they first become active, and/or their relative efficiency at various developmental stages, has been lacking. In this study, we evaluated these in four common Cre transgenic strains with thymus-initiated promoters (Lck, Cd2, or Cd4). We found that while all of them eventually labeled nearly all thymocytes, their kinetics were dramatically different, and other than Cd4[Cre], did not faithfully recapitulate the expression pattern of the corresponding endogenous gene. Perhaps even more importantly, while thymuses from some strains compared favorably to thymuses from control (Cre-negative) mice, we found that Cre expression could also result in off-target effects, including moderate to severe decreases in thymic cellularity. These effects occurred in the absence of loxP-flanked DNA target genes, and were dose and copy number dependent. Loss of cellularity was attributable to a specific decrease in CD4(+)8(+) immature cells, and corresponds to an increased rate of programmed cell death. In addition to a comprehensive analysis of activation kinetics in thymus-initiated Cre transgenes, our data show that Cre is toxic to CD4(+)8(+) cells in a dose-dependent fashion, and emphasize that the choice of thymus-initiated Cre strain is critically important for minimizing off-target effects of Cre.
The Scripps Research Institute, Jupiter, Florida, United States of America.
Jianjun
J
Shi
Howard T
HT
Petrie
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D018628:Gene Dosage; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D054503:Lymphoid Progenitor Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D013950:Thymus Gland / Q000166:cytology*; D019076:Transgenes / Q000235:genetics*
C045073:Cre recombinase; D019426:Integrases