Novel mouse alleles allow for sequential mutagenesis using the dual recombinase technology

Genetically engineered mouse models have been used extensively to study a wide variety of biological processes in vivo, and innovations in genetic engineering have made it possible to dissect more intricate biological questions. For example, the first mice that showed successful germline transmission of foreign DNA were created in the 1980s, and this allowed the generation of various transgenic “oncomice” such as the first mouse model of breast cancer using the MMTV-Myc transgene ¹, and the first mouse model of osteosarcoma using the MT-FosLTR transgene ². Subsequently in the early 1990s, mice were developed to have whole-body deletions of various genes such as the p53 tumor suppressor gene ³. The combination of the ability to introduce oncogenes and delete tumor suppressor genes through these initial transgenic mouse technologies contributed greatly to our current understanding of cancer development, progression, and treatment.

Nonetheless, transgenic mice and whole-body knock out mice sometimes do not survive development ⁴. To overcome this limitation, investigators harnessed site-specific recombinase systems from bacteriophages and yeasts such as the Cre-loxP and the Flp-FRT systems, which were first adapted to the mouse genome for germline transmission in the 1990s to enable temporally-regulated and tissue-specific genetic manipulations ^{5, 6}. Since then, many conditional mouse alleles utilizing transgenic or endogenous promoter-driven Cre and loxP-regulated genes have been generated to increase tissue-specificity of gene expression and decrease pre-mature lethality and other unwanted phenotypes. Additionally, replication-defective viruses (adenoviruses, lentiviruses) containing Cre recombinase (Adeno-Cre, Lenti-Cre) have also been used to further improve upon the temporal regulation of gene expression ⁷. Further modifications enabled the viruses to carry specific promoter-driven Cre recombinases to add tissue specificity after viral delivery ⁸. Finally, similar temporal regulation can be achieved by using fusion proteins combining Cre and mutated hormone receptors such as the estrogen receptor (Cre-ER^T2) ⁹. By utilizing this approach, metabolites of tamoxifen can be used to translocate Cre-ER^T2 to the nucleus and thus activate subsequent Cre-mediated gene modification.

Recently, there has been increasing interest in combining more than one recombinase system in the same mouse model using dual recombinase technology to allow for sequential mutagenesis and to better model sporadic cancers in the adult mouse ^{10, 11}. In our lab, we have generated two complimentary new mouse alleles that facilitates the regulation of Cre-ER^T2 by the Flp-FRT recombinase system. Both alleles are knocked into the endogenous Col1a1 locus for ubiquitous expression, and also at the same time reserve the two Rosa26 alleles for other transgenes of interest. In the first mouse allele, Col1a1^{FRT-Cre-ER-T2-FRT}, Cre-ER^T2 is flanked by FRT sites. The rationale to generate Col1a1^{FRT-Cre-ER-T2-FRT} mice is to enable whole animal ubiquitous expression of Cre-ER^T2 until exposure to Flp recombinase (Figure 1A). After Flp-mediated recombination of the FRT sites, cells are no longer able to express Cre-ER^T2 and therefore lose the ability to delete DNA flanked by loxP sites following exposure to tamoxifen. In this way, different mutations can be introduced in adjacent cells in vivo so that the consequences for intercellular interactions, such as cancer cells and stromal cells, can be studied.   In the second mouse allele, Col1a1^{FRT-STOP-FRT-Cre-ER-T2}, Cre-ER^T2 sits downstream of a FRT-flanked STOP cassette, which inhibits transcription of Cre-ER^T2. The rationale to generate Col1a1^{FRT-STOP-FRT-Cre-ER-T2} is that initially no cell expresses Cre-ER^T2 because transcription of Cre-ER^T2 is terminated by an upstream FRT site-flanked transcription STOP cassette (Figure 1B). However, after Flp-mediated recombination, the STOP cassette is excised. Therefore, these cells can initiate transcription of the Cre-ER^T2 fusion protein, which in response to subsequent exposure to tamoxifen translocates into the nucleus to recombine DNA flanked by loxP sites. Cells without exposure to Flp will not be able to undergo Cre-mediated DNA recombination. In this way, the Col1a1^{FRT-STOP-FRT-Cre-ER-T2} allele enables sequential mutations within the same cell over time. First, one mutation occurs in the cell from Flp recombinase, and then tamoxifen activates Cre recombinase in the same cell to mutate a second gene to study how the order of gene mutations may affect cellular outcome. In addition, multiple genes may be mutated by Flp recombinase to initiate tumor development. Then, the role of a loxP-flanked gene in tumor maintenance can be studied because only the tumor cell will express Cre-ER^T2. This allele can therefore be used to identify potential therapeutic targets.

Through characterization of the Col1a1^{FRT-Cre-ER-T2-FRT} mice using the reporter allele Rosa26^{mTmG 12}, we found that a single intraperitoneal injection of tamoxifen at 75mg/kg into Col1a1^{FRT-Cre-ER-T2-FRT}; Rosa26^mTmG/+ mice is sufficient to translocate the Cre-ER^T2 fusion protein into the nucleus and mediate recombination of the loxP-sites flanking tdTomato. This resulted in the deletion of the tdTomato red fluorescent protein, and the subsequent expression of eGFP green fluorescent protein from cells of all tissues examined. The limitation of this model is that there is an age- and tissue-dependent Cre-ER^T2 activation independent of tamoxifen administration, most notably in the pancreas and the liver. Therefore, in experiments involving these organs using the Col1a1^{FRT-Cre-ER-T2-FRT} mice, age of the mice at the onset of Flp and tamoxifen administration is crucial for the interpretation of the results.

We also characterized the Col1a1^{FRT-STOP-FRT-Cre-ER-T2} mice using the reporter allele Rosa26^mTmG. When Col1a1^{FRT-STOP-FRT-Cre-ER-T2}; Rosa26^mTmG/+ mice were given a single intraperitoneal injection of tamoxifen at 75mg/kg, there was no Cre-ER^T2-mediated deletion of the tdTomato red fluorescent protein or expression of the eGFP green fluorescent protein. Next, we generated a mouse model of soft tissue sarcoma using the new allele to verify the ability of the STOP cassette to be removed by Flp, and the subsequent ability of Cre-ER^T2 to manipulate loxP-flanked alleles. In this model, Col1a1^{FRT-STOP-FRT-Cre-ER-T2}; Kras^{FRT-STOP-FRT/+}; p53^FRT/FRT; Rosa26^mTmG/+ mice were first administered intramuscular injection of adenovirus carrying Flp recombinase in the hindlimb to form soft tissue sarcomas at the site of injection via the expression of the oncogenic KRAS protein and elimination of the p53 tumor suppressor protein ^{11, 13, 14}. Following the formation of soft tissue sarcomas, either single intratumoral injection of 4-hydroxytamoxifen or several doses of systemic 4-hydroxytamoxifen were administered. The resulting tumor showed tumor parenchymal cells with deletion of tdTomato red fluorescent protein and expression of eGFP green fluorescent protein, while the tumor stromal cells such as the vasculature continued to express tdTomato. One potential challenge in this model is that while the Col1a1^{FRT-STOP-FRT-Cre-ER-T2} is functional, single intraperitoneal injection of tamoxifen failed to activate Cre-ER^T2 in primary sarcomas. Therefore, the delivery of an adequate level of tamoxifen and/or its metabolites into the tumor is critical to activate Cre-ER^T2 expression with this allele.

We anticipate that these two new alleles will allow for increased control of gene manipulation in genetically engineered mouse models. These alleles, in conjunction with other new technologies in the field such as the CRISPR/Cas9 system ¹⁵, have the potential to bring together the speed and efficiency of the CRISPR/Cas9 system with the spatial and temporal control of dual recombinase technology, manipulate the genome in vivo to study development, cancer and other diseases in the mouse.

Minsi Zhang and David Kirsch, Department of Radiation Oncology, Duke University Medical Center

Main reference

Zhang, M., & Kirsch, D. The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis. Disease Models & Mechanisms. 2015. 8(9), 1155-1166 DOI: 10.1242/dmm.021204

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