Perspective: The current state of Cre driver mouse lines in skeletal research: Challenges and opportunities

Abstract

The Cre/Lox system has revolutionized the ability of biomedical researchers to ask very specific questions about the function of individual genes in specific cell types at specific times during development and/or disease progression in a variety of animal models. This is true in the skeletal biology field, and numerous Cre driver lines have been created to foster conditional gene manipulation in specific subpopulations of bone cells. However, as our ability to scrutinize these models increases, an increasing number of issues have been identified with most driver lines. All existing skeletal Cre mouse models exhibit problems in one or more of the following three areas: (1) cell type specificity-avoiding Cre expression in unintended cell types; (2) Cre inducibility-improving the dynamic range for Cre in inducible models (negligible Cre activity before induction and high Cre activity after induction); and (3) Cre toxicity-reducing the unwanted biological effects of Cre (beyond loxP recombination) on cellular processes and tissue health. These issues are hampering progress in understanding the biology of skeletal disease and aging, and consequently, identification of reliable therapeutic opportunities. Skeletal Cre models have not advanced technologically in decades despite the availability of improved tools, including multi-promoter-driven expression of permissive or fragmented recombinases, new dimerization systems, and alternative forms of recombinases and DNA sequence targets. We review the current state of skeletal Cre driver lines, and highlight some of the successes, failures, and opportunities to improve fidelity in the skeleton, based on successes pioneered in other areas of biomedical science.

Keywords: Cre; Floxed allele; LoxP; Recombination; Skeletal models.

Figure 1:

Schematic overview of the Cre/Lox system for recombining genomic DNA. A typical gene of interest (GOI) is depicted, with hypothetical exons shown in grey boxes and the intervening introns shown as black lines. 34bp LoxP sequences introduced into the 1^st and 3^rd introns are indicated by orange triangles, but their presence does not affect the messenger RNA molecule, as indicated by the identical transcript shown for WT and floxed GOI. (B) Nucleotide-level view of the 5’ loxP site, indicating the 13bp inverted palindromic Cre recognition sites, as well as the intervening spacer which provides directionality and is the site for restriction activity. The lower panel shows a pair of Cre molecules binding to the recognition sites, with idealized extensions cutting after the first base (in each direction) of the spacer region. (C) Enzymatically cleaved LoxP site, opened up to show the 6bp overhang created, which will ultimately ligate with the complementary overhang from the 3’ loxP site after the intervening piece is removed.

Figure 2:

Idealized depiction of the tet-off system for driving Cre expression in osteoclasts. The lower left image shows the Cre transgene, which is driven by the tetO.CMV promoter. The tetO.CMV promoter requires 2 components in order to transcribe Cre. The first is the presence of a specialized transcription factor known as tTA, and the second is the absence of the antibiotic doxycycline in order to allow tTA to be active. The upper panel shows tTA expression being driven by the cathepsin-K promoter, which gives cell selectivity (osteoclast) to the system. The lower right panel shows the presence of tTA, and the absence of dox, which meets the permissive requirements for tetO.CMV activation and Cre transcription. The Ctsk promoter driving tTA expression provides spatial (cell type) control of Cre, while the removal of dox from the diet provides temporal control of Cre. As mentioned in the text, reverse tet systems are also used (tet-on), where dox activates rather than inhibits tTA.

Figure 3:

Three doses of tamoxifen in Sost-CreERt2 mice crossed onto the Ai9-tdTomato reporter reveals a poor dynamic range (compare top induced panel with middle uninduced panel). The tdTomato reporter has no detectable activity in Cre-negative mice (lower panel).

Figure 4:

(A) Venn diagram indicating the expression characteristics for two well-known osteocyte genes—Sost and Mepe. Both exhibit expression outside of the osteocyte population, but their extra-osteocytic expression profiles do not overlap. (B) Splitting the Cre molecule into two components and driving each component by different promoters (in this case, using Sost and Mepe as hypothetical drivers) allows expression of both Cre components only in cells where Mepe and Sost expression overlap—i.e., in the osteocytes. The components are able to reassemble and gain activity by designing the Cre sequences such that the two resulting protein fragments are attached to protein splicing elements known as inteins. Complementary inteins attract to one another and enact a series of autocatalytic peptide bone rearrangements that induces self-excision of the intein components and ligation of the flanking polypeptides. (C) In cells that express Sost but not Mepe (for example, coronary artery smooth muscle cells), the c-terminal fragment of Cre alone is expressed, but without the complementary n-terminal fragment, no active Cre is produced. In cells that express Mepe but not Sost (for example, cerebral cortex neurons), the n-terminal fragment of Cre alone is expressed, but without the complementary c-terminal fragment, no active Cre is produced. In cells that express both Sost and Mepe (i.e., osteocytes), both n- and c-terminal Cre fragments are expressed, and the associated inteins self-assemble the Cre fragments and auto-excise the intein, rendering active Cre protein only in osteocytes.

Figure 5:

Next-generation inducible Cre systems to achieve greater inducibility, lower leakiness, and reduced off-target effects (e.g., tamoxifen effects on bone) in the cell type of interest. (A) The two-promoter approach is explained in Fig 3, but here it is assumed to drive Cre fragments that are fused to dimerization elements. For sake of example, the Cre protein is split between amino acids 270 and 271 such that the n-terminal contains aa1-270 and the c-terminal fragment contains aa271-334. Two dimerization systems are depicted; on the left is the GAI/GID1 system which dimerizes via exposure to gibbirelin (GIB)and brings the two Cre fragments into a configuration where they can ligate and induce recombination activity. On the right is the rapamycin (RAPA)-sensitive FKBP/FRB system, which functions similarly to the GAI/GID1 system. (B) Inducible system using degraded Cre-ecDHFR fusion protein (ddCre). ddCre is rapidly degraded by the proteosome, rendering it useless to recombine LoxP sites. ddCre is stabilized by the addition of the antibiotic analog trimethoprim (TMP), which induces ddCre survival and results in Cre activity.

Figure 6:

Self-inactivating Cre systems have been designed and implemented in other model organisms, but not in mice. Self-inactivation can be achieved by flanking the Cre-producing sequence with LoxP sites, such that both the gene of interest (GOI) and the Cre sequence are recombined away soon after Cre turns on, leaving a lonely LoxP site at each locus. This self-limiting approach is likely to reduce the toxic effects of sustained Cre expression, which serves no purpose once LoxP is recombined.