Activation Kinetics and Off-Target Effects of Thymus-Initiated Cre Transgenes

PLoS One

Jianjun Shi¹  Howard T. Petrie¹ 

[1] The Scripps Research Institute, Jupiter, Florida, United States of America

关键词: Thymocytes;    Thymus;    Cell death;    Cell differentiation;    Apoptosis;    Flow cytometry;    Gene expression;    Cell staining;   

DOI  :  10.1371/journal.pone.0046590

学科分类：医学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY