2026-03-31 理化学研究所
「遺伝子発現の抑制漏れ」を最小限に抑えた遺伝子改変の新手法
＜関連情報＞
- https://www.riken.jp/press/2026/20260331_1/index.html
- https://www.cell.com/cell-reports-methods/fulltext/S2667-2375(26)00069-X
高感度生物発光レポーターを用いたマウスゲノムにおけるCre依存性DNA構築物の漏出発現の評価 Assessing leaky expression of Cre-dependent DNA constructs in the mouse genome using sensitive bioluminescent reporters
Toshiaki Nakashiba ∙ Takashi Sugiyama ∙ Satoshi Iwano ∙ … ∙ Atsushi Yoshiki ∙ Atsushi Miyawaki ∙ Kuniya Abe
Cell Reports Methods Published:March 30, 2026
DOI:https://doi.org/10.1016/j.crmeth.2026.101369
Motivation
The conditional expression of genes in model organisms using the Cre-loxP system is a key strategy for deciphering gene functions at the organismal level. However, research on leaky gene expression in Cre-dependent DNA constructs—even in the absence of Cre recombinase—remains limited, despite the potential of misinterpreting experimental data. In the present study, a highly sensitive bioluminescence reporter was employed to explore more effective strategies to prevent such leakage. Consequently, the suppression of leaky gene expression led to the development of a reporter mouse strain that enables comprehensive whole-body mapping of Cre recombination sites.
Highlights
- Cre-independent leaky expression is detected and quantified using sensitive AkaBLI
- A transcriptional STOP cassette alone is insufficient to prevent leaky expression
- Dual transcriptional and translational controls effectively suppress leakage
- Leak-suppressed reporter mice enable whole-body mapping of Cre recombination sites
Summary
Leaky expression of genes in Cre-dependent DNA constructs in the absence of Cre recombinase has the potential to undermine precision of conditional gene manipulation. Here, we reliably detected and quantified this phenomenon in mice using highly sensitive bioluminescence imaging (BLI). By generating a series of knockin mice carrying Cre-dependent DNA constructs incorporating AkaBLI system, we found that a transcriptional STOP cassette alone was insufficient, producing significant Cre-independent leaky expression. This leaky expression can be suppressed by a combination of transcriptional and translational controls, facilitating precise detection and quantification of Cre-mediated recombination. Furthermore, application of leak-suppressed AkaBLI reporter mice to Cre-driver mice led to unambiguous identification of Cre-mediated recombination at intended and unintended sites throughout the mouse body. Thus, our data provide a generalizable solution to the leaky expression associated with Cre-loxP system, thereby enabling generation of diverse model organisms that require tight gene expression control.
