The a"MAZE"ing world of lung-specific transgenic mice

Abstract

The purpose of this review is to give a comprehensive overview of transgenic mouse lines suitable for studying gene function and cellular lineage relationships in lung development, homeostasis, injury, and repair. Many of the mouse strains reviewed in this Perspective have been widely shared within the lung research community, and new strains are continuously being developed. There are many transgenic lines that target subsets of lung cells, but it remains a challenge for investigators to select the correct transgenic modules for their experiment. This review covers the tetracycline- and tamoxifen-inducible systems and focuses on conditional lines that target the epithelial cells. We point out the limitations of each strain so investigators can choose the system that will work best for their scientific question. Current mesenchymal and endothelial lines are limited by the fact that they are not lung specific. These lines are summarized in a brief overview. In addition, useful transgenic reporter mice for studying lineage relationships, promoter activity, and signaling pathways will complete our lung-specific conditional transgenic mouse shopping list.

Figure 1.

Schematic of Cre and Tet transgenic systems. (A) Cre, or CreER, is expressed from a cell or tissue-specific promoter. (i) Cre is active in every cell in which it is expressed. Levels of activity depend on expression levels. (ii) CreER is not active (it is actively excluded from the nucleus) until tamoxifen is administered. This allows for temporal control of the system and reduces nonspecific effects of the Cre. (B) Cre catalyzes recombination between its recognition (LoxP) sites. This results in a permanent genetic rearrangement, which is inherited by daughter cells. The most common arrangement of LoxP sites are (i) flanking a crucial exon of an endogenous gene to mediate loss of function or (ii) flanking a transcriptional STOP to mediate reporter gene expression and/or overexpression. In this case, a ubiquitous promoter is usually used to drive the gene of interest (a reporter, coding sequence, or hairpin), but the transcript is disrupted by the LoxP-flanked transcriptional STOP. After Cre-mediated deletion of the stop, the gene of interest is permanently expressed. Such cassettes are frequently targeted to the ubiquitously expressed ROSA26 locus for example (18). (C) The tetracycline (Tet) system has two versions, Tet-on and Tet-off. (i) Tet-on. The reverse tetracycline transactivator (rtTA) is expressed from a cell or tissue-specific promoter. In the Tet-on system, administration of doxycycline (Dox) activates the responsive transgene. rtTA is not functional until Dox is present. Upon Dox binding, rtTA binds to its tetO recognition sites and activates transcription. The system is reversible. Once Dox has been metabolized, rtTA no longer binds to the tetO sites and the transgene turns off. Similarly, the transgene is turned off in any descendents of the cell that no longer express rtTA. (ii) Tet-off. The tetracycline transactivator (tTA) is expressed from a cell- or tissue-specific promoter. In the Tet-off system, the tetO-responsive transgene is constitutively active, and administration of Dox represses the transgene. Dox binds to tTA, which allows it to bind to the tetO elements and suppress transcription. The Tet-off system is also reversible. (D) Combining the Cre and Tet systems. (i) Dox-conditional Cre activation. rtTA is expressed from a cell- or tissue-specific promoter, and Cre is placed under the control of the tetO elements. Upon Dox administration, Cre is transcribed and enters the nucleus to recombine its LoxP target sites. In this case, deletion or activation of the LoxP-flanked target gene is permanent. Any off-target effects of the Cre can be reduced by minimizing the amount of Dox administered. However, Dox treatment itself can also affect lung development and homeostasis, and this must also be controlled for. (ii) Cre-conditional rtTA activation. Cre is expressed from a cell- or tissue-specific promoter, and rtTA is placed upstream of a LoxP-flanked STOP cassette. Cre activity results in constitutive expression of rtTA in the Cre-expressing cells and all of their descendants. However, the tetO-responsive transgene is only transcribed in the presence of Dox. In this case, activation of the gene of interest is reversible.