The generation and characterization of novel Col1a1 and Col1a1 FRT-Cre-ER-T2 mice for sequential mutagenesis

Novel genetically engineered mouse models using the Cre-loxP or the Flp-FRT systems have generated useful reagents to manipulate the mouse genome in a temporally-regulated and tissue specific manner. By incorporating a constitutive Cre driver line into a mouse model in which FRT-regulated genes in other cells types are regulated by Flp-FRT recombinase, gene expression can be manipulated simultaneously in separate tissue compartments. This application of dual recombinase technology can be used to dissect the role of stromal cells in tumor development and cancer therapy. Generating mice in which Cre-ER is expressed under Flp-FRT-mediated regulation would enable step-wise manipulation of the mouse genome using dual recombinase technology. Such next-generation mouse models would enable sequential mutagenesis to better model cancer and define genes required for tumor maintenance. Here, we generated novel genetically engineered mice that activate or delete Cre-ER in response to Flp recombinase. To potentially utilize the large number of Cre-loxP regulated transgenic alleles that have already been targeted into the Rosa26 locus, such as different reporters and mutant genes, we targeted the two novel Cre-ER alleles into the endogenous Col1a1 locus for ubiquitous expression. In the Col1a1 mice, Flp deletes Cre-ER, so that Cre-ER is only expressed in cells which have never expressed Flp. In contrast, in the Col1a1 mice, Flp removes the STOP cassette to allow Cre-ER expression so that Cre-ER is only expressed in cells that previously expressed Flp. These two new novel mouse strains will be complementary to each other and will enable the exploration of complex biological questions in development, normal tissue homeostasis, and cancer. D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t INTRODUCTION Genetically engineered mouse models are ideal for studying complex mammalian biological processes in vivo. Advances in genetic engineering have enabled the development of increasingly more precise, controlled manipulation of the mouse genome so that more intricate biological questions can be explored. Site-specific recombinase systems, such as the Cre-loxP and the Flp-FRT systems, were developed to allow for temporally-regulated and tissue-specific gene modification (Dymecki, 1996; O'Gorman et al., 1991; Orban et al., 1992; Sauer and Henderson, 1988). A large number of conditional mouse alleles utilizing the CreloxP system have been generated. These include various loxP flanked genes and a number of endogenous promoter driven Cre recombinase alleles that are constitutively expressed. Cre recombinase expressed from endogenous promoters enables tissue specific deletion of DNA flanked by loxP sites to alter gene expression. The resulting tissue-specificity for recombination of the loxP sites decreases the likelihood of embryonic lethality. For example, Villin-Cre (el Marjou et al., 2004), which is expressed in the entire intestinal and colonic epithelium, and FapblCre (Saam and Gordon, 1999), which is expressed in the distal 2/3 of the small intestine and the entire colonic epithelium, can be used to express the embryonically lethal oncogenic KRAS by recombining Kras (Jackson et al., 2001) to generate diffuse colonic hyperplasia in mice (Haigis et al., 2008). However, gene modification in the embryo may not fully recapitulate gene manipulation in the adult to model somatic gene mutations that lead to the development of sporadic cancers. One way to circumvent this limitation is to exogenously deliver Cre recombinase to the adult mice via infection with replicative-deficient adenoviruses (Adeno-Cre) (Wang et al., 1996). For example, intratracheal delivery of Adeno-Cre to adult mice with Rb and p53 mutations (Marino et al., 2000) generated primarily small cell lung tumors that closely D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t resembled human small cell lung cancer (Meuwissen et al., 2003). In addition to small cell lung tumors, this method also generated lung adenocarcinomas as well as medullary thyroid carcinomas both in mice that also developed small cell lung tumors and in mice that did not. This drawback to using viral vectors, particularly in cancer biology, is due to the fact that many cell types can be infected by the adenovirus. Thus, the cancer-initiating cell (i.e. cellof-origin) can vary between tumors from different animals as well as different tumors within the same animal. This heterogeneity may lead to phenotypic differences in these mouse models of cancer. To further limit expression of Cre to specific cell types within an organ, Cre can be expressed from the adenovirus using a cell-type specific promoter (Sutherland et al., 2011; Sutherland et al., 2014). Innovations in site-specific recombinase systems continue to improve tissue specificity and temporal regulation of gene expressions. For example, fusion proteins consisting of Cre recombinase and a mutated estrogen receptor that preferentially binds to the metabolites of the estrogen analog tamoxifen, such as Cre-ER, have been generated (Indra et al., 1999). These fusion proteins allow for tamoxifen-inducible Cre recombinase to translocate into the nucleus and modify the genome. These Cre-fusion proteins allow temporal activation of oncogenes and/or deletion of tumor suppressor genes in the adult mouse in a cell-type specific manner to generate cancer (Blum et al., 2013). Recently, a second site-specific recombination system, Flp-FRT, which originated from S. cerevisiae, has been applied to study cancer. Similar to Cre, Flp recombinase recognizes 34bp sequences of DNA termed FRT sites. Like Kras mice, investigators have generated mice in which Kras is regulated by a transcriptional terminator sequence flanked by FRT sites in the endogenous promoter (Kras) (Young et al., 2011). Similar to p53 mice, we previously generated mice with the DNA binding domain of p53 flanked by FRT sites (p53) (Lee et al., 2012). When the p53 D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t alleles are combined with Kras mice, Flp can generate primary sarcomas, lung cancer, and pancreatic cancer (Lee et al., 2012; Moding et al., 2015; Moding et al., 2014; Schonhuber et al., 2014). The use of only one recombinase system to mutate multiple genes limits the scope of cancer research because all genetic manipulations are completed at the same time. In the case of mutations that drive cancer, the use of a single recombinase system prohibits the study of sequential mutagenesis to determine how the order of different driver gene mutations affects tumor phenotypes. Moreover, the consequence of a gene mutation at tumor initiation may be different from a mutation that is acquired later during tumor development. Indeed, when a single recombinase system is used to mutate multiple genes simultaneously, it is possible that cells may adapt to the loss of a gene at tumor initiation in ways that are different than if the same gene had been mutated later during tumor development. This may be particularly important for genetic experiments designed to identify therapeutic targets, which are required for cancer maintenance because in certain cases, inhibition of a target at the time of cancer initiation may lead to a different outcome than when a target is inhibited in an established tumor. Others have demonstrated that the Flp-FRT and the Cre-loxP systems can be used sequentially. For example, lung adenocarcinoma has been generated by sequentially delivering Flp recombinase by adenovirus and Cre recombinase either by adenovirus or by tamoxifen to activate Cre-ER in mice harboring Braf FRT-STOP-FRT-V600E and either p53 or Cdkn2a mutations (Shai et al., 2015). Likewise, pancreatic adenocarcinoma has been generated by sequential mutagenesis, where first Kras and Rosa26 Cre-ER-T2 are recombined by Pdx1-Flp to generate pancreatic tumors harboring oncogenic KRAS and the Cre-ER fusion protein, then tamoxifen is delivered to the animal to activate D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t Cre-ER to recombine p53 in the pancreatic tumors, resulting in pancreatic tumors with both KRAS and p53 mutations (Schonhuber et al., 2014). Here, we generated novel genetically engineered mice that activate or delete Cre-ER in response to Flp recombinase. Specifically, we generated two new mouse strains in which Cre-ER is knocked into the endogenous Col1a1 locus so that the Rosa26 locus can be used for other genes or reporters of interest. In one allele, Col1a1, Cre-ER is flanked by FRT sites. In the other allele, Col1a1, Cre-ER is regulated by a FRT-STOP-FRT cassette. In Col1a1 mice, cells will only express Cre-ER and have the capacity for tamoxifen-mediated recombination of loxP sites if they have never had prior exposure to Flp recombinase. Therefore, this strain will be useful for studying genes in stromal cells of established tumors that are initiated by Flp. In contrast, in Col1a1 mice in which Cre-ER is downstream from a FRT-STOP-FRT cassette, cells will only express Cre-ER and have the capacity for tamoxifen-mediated recombination of loxP sites after Flp-mediated removal of the STOP cassette. Therefore, this strain will be useful for sequential mutagenesis within tumor cells or for modifying the tumor cell genome in established cancers. Thus, these two new mouse strains will be complimentary to each other and will enable the exploration of complex biological questions in cancer, normal development, and tissue homeostasis. RESULTS Generation of Flp-FRT regulated Cre-ER alleles In order to develop technology for sequential mutagenesis in vivo using two sitespecific recombinase systems, we generated Col1a1 and Col1a1 ER-T2 mice. The rationale to generate Col1a1 mice was to enable whole animal ubiquitous expression of Cre-ER until exposure to Flp recombinase (Figure 1A). After FlpD ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t mediated recombination of the FRT sites, cells are no longer able to express Cre-ER 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. The rationale to generate Col1a1 was that initially no cell expresses Cre-ER because transcription of Cre-ER is terminated by an upstream FRT siteflanked 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 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 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. This allele can therefore be used to identify potential therapeutic targets. To generate mice in which the expression of Cre-ER is regulated by the Flp-FRT recombinase system and to fully utilize the large number of Cre-loxP regulated transgenic alleles in the Rosa26 locus, such as fluorescent reporters and mutant genes, the two novel Cre-ER alleles were targeted into the endogenous Col1a1 locus for ubiquitous expression (Figure 2A-B). Targeting constructs for the generation of Col1a1 and Col1a1 mice consisted of sequence from the Col1a1 genomic DNA, CAG promoter, Cre-ER regulated by FRT sites, and a neomycin selection cassette flanked by D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t attP and attB sites. Therefore, transcription of the targeted Cre-ER recombinase will be driven from the endogenous Col1a1 locus and enhanced by the addition of a CAG promoter. Constructs of the two Cre-ER alleles were electroporated into 129/SVJae ES cells and successfully targeted ES cells were selected by neomycin (G418) treatment. Positively selected ES cells were analyzed for successful homologous recombination by Southern Blot of genomic DNA (Figure 2C-D). Correctly targeted ES clones were injected into C57BL/6 blastocysts and male high-percentage chimeras were selected to breed with C57BL/6 females to identify germline transmission of the Cre-ER alleles. The neomycin selection cassettes were removed by PhiC31 integrase-mediated recombination of the attP and attB sites in vivo by crossing the mice to the pre-existing Rosa26 strain (Raymond and Soriano, 2007). Germline transmission of Col1a1 was verified by PCR for the construct-specific neomycin selection cassette on tail-tip DNA (Figure 3A). Heterozygosity for the knock-in allele was demonstrated by PCR for the wildtype Col1a1 locus (Figure 3A). Successful PhiC31-mediated excision of the neomycin selection cassette was verified by PCR for gene products specific to the recombined neomycin selection cassette (excised NEO), the unrecombined neomycin selection cassette (NEO), the wildtype Col1a1 locus, and the mutant PhiC31 allele (Figure 3B). Germline transmission of a single copy of Col1a1 was verified by PCR for the construct-specific STOP cassette, the neomycin selection cassette, and the wildtype Col1a1 locus on tail-tip DNA (Figure 3C). Deletion of the neomycin selection cassette by PhiC31 was verified by PCR for gene products specific for the recombined neomycin selection cassette (excised NEO), the unrecombined neomycin selection cassette (NEO), the STOP cassette, the wildtype Col1a1 locus, and the mutant PhiC31 allele (Figure 3D). One founder mouse for each of the novel Cre-ER strains was used to propagate the colony. D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t Characterization of Flp-FRT regulated Cre-ER mice To examine Cre-mediated recombination at a cellular level, tissues from Col1a1 ; Rosa26 mice were examined by immunofluorescence with and without tamoxifen treatment (Figure 4). Without Cre-mediated recombination, the Rosa26 allele transcribes only the tdTomato fluorescent protein. After Cre-mediated excision of tdTomato, eGFP is expressed from the same locus. Because these mice are heterozygous for the Rosa26 allele, any expression of eGFP indicates Cre-mediated excision of tdTomato. In tissues taken from 6-week-old mice (n=2) without tamoxifen treatment (Figure 4A), expression of tdTomato was widespread, and there is minimal tissue-specific eGFP expression. Tissues with some eGFP expression include the pancreas (Figure 4Avii), and to a lesser extent a few skeletal muscle fibers (Figure 4Aiii). When tissues from 6-month-old mice (n=3) obtained 30 days after treatment with a single dose of 75mg/kg tamoxifen in corn oil by intraperitoneal injection were examined (Figure 4B), there was widespread eGFP expression and minimal tdTomato retention. The tissue with highest tdTomato retention was the brain (Figure 4Bxxxii), where there was diffuse low tdTomato expression with regions of high eGFP expression (Figure 4Bxxxi). This may reflect diffusion limitations of tamoxifen metabolites across the blood-brain barrier or brain-specific expression from the Col1a1 locus, synthesis of functional eGFP protein, and degradation of tdTomato. The lower expression of tdTomato in the brain tissue of Col1a1; Rosa26 mice treated with tamoxifen as compared to untreated controls (Figures 4Axxxii) suggests the rate of degradation of tdTomato plays a greater role. Additionally, it appears that certain cell types do have higher tdTomato or higher eGFP expression than others, which may be due to the activity of the Rosa26 locus or perhaps a consequence of the way the tissue is affected by processing. For example, the endothelial cells of the brain (Figure 4Axxxii) have higher tdTomato expression than the neurons. The glomeruli of the kidney (Figure 4xx) have higher D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t tdTomato expression than the collecting ducts. Overall, the lung parenchyma seems to have lower tdTomato expression (Figure 4Axii) than other tissues such as the brain (Figure 4Axxxii). To determine the activity of Cre-ER in Col1a1; Rosa26 mice in the absence of tamoxifen, tissues from 4-month-old (n=2) and 6-month-old mice (n=3) without tamoxifen treatment were examined (Figure 5). The tissues with the most widespread eGFP expression without tamoxifen treatment in the 6-month-old mice include the pancreas (Figure 5Bvii), the liver (Figure 5Bxv), and the skeletal muscle (Figure 5Biii). There is timedependent tissue-specific increase in eGFP expression when comparing the 4-month-old and 6-month-old mice (Figure 5A-B). For example, the skeletal muscle fibers in the 4-month-old mice (Figure 5Aiii) appear to have less eGFP expression than those in the 6-month-old mice (Figure 5Biii). Additionally, the pancreas of the 4-month-old mice (Figure 5Avii) appears to have less eGFP expression than the pancreas of the 6-month-old mice (Figure 5Bvii). The liver of both the 4-month-old (Figure 5xv) and the 6-month-old mice (Figure 5Bxv) appear to have similar eGFP expression. In contrast, the brain has no eGFP expression with the exception of a subset of the vasculature in both the 4-month-old mice (Figure 5Axxxi) and the 6-month-old mice (Figure 5Bxxxi). Taken together, these results demonstrate that there is time-dependent and tissue-specific Cre-ER activity in Col1a1 mice in the absence of tamoxifen, with the brain tissue being the least affected organ, and the pancreas, liver, and skeletal muscle fibers being the most affected organs. In addition, tamoxifen potently induces Cre-ER-mediated recombination of unrecombined loxP sites. To examine the leakiness of Cre-ER in Col1a1 mice, tissues from 6-month-old Col1a1; Rosa26 mice were examined by immunofluorescence with (n=2) and without (n=2) tamoxifen treatment (Figure 6). In the absence of tamoxifen, there was widespread tdTomato expression without evidence of eGFP D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t expression in any of the tissues examined (Figure 6A). When tissues were collected 30 days after intraperitoneal delivery of one dose of 75mg/kg tamoxifen in corn oil, there continued to be widespread tdTomato expression without evidence of eGFP expression in any of the tissues examined (Figure 6B). These results indicate that the STOP cassette is functional and does not allow for Cre-ER-mediated transcription in the absence of Flp-mediated recombination and excision of the STOP cassette. To determine if the STOP cassette for the Col1a1 allele can be excised by Flp-mediated recombination in vivo, mice with the Col1a1 allele were crossed to Kras; p53 mice (Lee et al., 2012) to generate Col1a1; Kras; p53 mice. Adenovirus expressing mammalian optimized Flp recombinase (Adeno-FlpO) was injected into the hindlimb of these mice to generate primary soft tissue sarcomas. Once sarcomas developed, the bulk tumor tissues were excised and dissociated into single cell suspensions and cultured in vitro. Genomic DNA was isolated from these cells and the recombination of the STOP cassette was verified by PCR (Figure 7A). Tail DNA taken at time of genotyping from a Col1a1 T2-FRT mouse was used as a positive control for the absence of STOP cassette, and tail DNA taken at time of genotyping from a Col1a1 mouse was used as a negative control for the recombined STOP cassette. To examine the functionality of Col1a1 allele in a tumor model, these mice were crossed to Kras; p53; Rosa26 mice to generate Col1a1;Kras; p53;Rosa26 mice. Primary sarcomas were generated in the hindlimb of these mice by intramuscular injection of AdenoFlpO. After the initial tumor was palpated in the Col1a1; Kras ; p53; Rosa26 mice, a single dose of 75mg/kg tamoxifen in corn oil was delivered via intraperitoneal injection. Tumors (n=2) were collected 10 days after the D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t tamoxifen delivery, and immunofluorescence was used to evaluate tdTomato and eGFP expression (Figure 7B). Unexpectedly, the whole tumor tissue remained tdTomato positive without any evidence of eGFP expression. Because the STOP cassette can be appropriately removed by FlpO as shown in Figure 7A, the lack of tdTomato deletion and eGFP expression may be due to inadequate levels and/or penetration of the active tamoxifen metabolite into the tumor tissue. Therefore, alternative approaches of tamoxifen delivery were explored in subsequent cohorts. After the initial tumor was palpated in a second cohort (n=2) of Col1a1;Kras; p53;Rosa26 mice, a single dose of 0.75mg 4-hydroxytamoxifen in DMSO was delivered via intratumoral injection. Tumors were collected 10 days after the 4-hydroxytamoxifen delivery, and tdTomato and eGFP expression were evaluated by immunofluorescence (Figure 7C). In contrast to tumors from mice that were given intraperitoneal tamoxifen injection, the bulk tumor from mice that received intratumoral 4-hydroxytamoxifen were mostly eGFP positive with scattered tdTomato expression. For another set (n=2) of Col1a1;Kras ; p53;Rosa26 mice, a single dose of 2mg 4-hydroxytamoxifen in PBS was delivered via subcutaneous injection between the scapula. Twenty-four hours following the single injection, the tumors were harvested, and tdTomato and eGFP expression were evaluated by immunofluorescence (Figure 7D). These tumors demonstrated areas of small clusters of cells that were eGFP positive, while the majority of the tumors remained tdTomato positive. This demonstrates that a single high dose of 4-hydroxytamoxifen delivered systemically can penetrate the bulk tumor, but only to a limited area that is likely well perfused. Finally, after the initial tumor was palpated in the lower limb in a Col1a1 ; Kras; p53; Rosa26 mouse, one dose of 2mg 4hydroxytamoxifen in PBS was delivered via intraperitoneal injection. Twenty-four hours following the first injection, 2mg of 4-hydroxytamoxifen in PBS was delivered via D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t subcutaneous injection between the scapula. Two more subcutaneous injections of 4hydroxytamoxifen were delivered at the same site every 24 hours for a total of 4 doses (1 intraperitoneal and 3 subcutaneous). This tumor was collected 1 day after the last dose of 4hydroxytamoxifen, and tdTomato and eGFP expression were evaluated by immunofluorescence (Figure 7E). This tumor demonstrated scattered areas with high eGFP expression and larger areas with diffusely lower eGFP expression, and scattered tdTomato expression. This suggests that administration of multiple high doses of systemic 4hydroxytamoxifen is capable of penetrating the tumor tissue to enable Cre-ER-mediated recombination of loxP sites. Taken together, these results show that the Cre-ER in both Col1a1 and Col1a1 mice is functional and that the recombinase activity in both mice can be potently induced by the active tamoxifen metabolite. Additionally, the expression of Cre-ER in Col1a1 mice is tightly controlled by the STOP cassette. However, in this primary sarcoma model, penetration of the active tamoxifen metabolite into the tumor following a single intraperitoneal tamoxifen injection is insufficient to activate the Col1a1 allele. The limited penetration of the active tamoxifen metabolite after a single intraperitoneal injection of tamoxifen may be due to the large size (approximately 200mm) of the sarcomas at time of palpation and thus may not be a limitation for other tumor models where the tumors are smaller. Even in the primary sarcoma model, the limited penetration following a single intraperitoneal injection of tamoxifen can be overcome by several doses of 4-hydroxytamoxifen delivered by intraperitoneal and subcutaneous routes. Finally, in the Col1a1 mice there is a time-dependent recombinase activity of Cre-ER in the absence of tamoxifen exposure, which may limit its utility in certain tissues for experiments that would require waiting several months before the administration of tamoxifen. D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t DISCUSSION The primary mouse model is an important tool to study biological processes in vivo; therefore, novel mouse strains and mouse models provide an opportunity for scientific advancement. Here, we have generated two novel mouse strains to facilitate more precise manipulation of the mouse genome to answer complex questions. The two novel mouse strains take advantage of different site-specific recombinase systems to enable sequential mutagenesis separated in time and cellular location. These novel mouse strains will facilitate the study of complex questions in development, normal tissue homeostasis, and disease processes, such as cancer. For example, these strains have a number of advantages for studying the role of genes in either tumor cells or stroma. First, the knock-in alleles of both novel mouse strains are targeted to the ubiquitously expressed Col1a1 locus instead of the frequently used Rosa26 locus. This Col1a1 site was selected with the available Rosa26 knock-in alleles in mind. An advantage of knock-in alleles over other transgenic approaches that generate alleles by random integration into the mouse genome is that the precise location of the knock-in allele is known. However, each mouse can only harbor two different copies of a mutant gene at one locus. As there are many Rosa26 knock-in alleles ranging from mutant genes to fluorescent reporters, the placement of the two novel Flp-regulated Cre-ER alleles at the Col1a1 locus retains the availability of both alleles of the Rosa26 locus for other genes-of-interest. Our results indicate that the Col1a1 locus is a good alternative to the Rosa26 locus for ubiquitous gene expression as noted by diffuse eGFP expression after tamoxifen-mediated deletion of tdTomato in Col1a1; Rosa26 mice. In addition, Cre-ER is tightly regulated by the STOP cassette in Col1a1; Rosa26 mice, and after successful removal of the STOP cassette by Adeno-FlpO, Cre-ER can be efficiently D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t activated by 4-hydroxytamoxifen in primary soft tissue sarcomas via intratumoral injection or multiple systemic injections to express eGFP in the tumor parenchyma. A limitation of the Col1a1 mice is the age-dependent tissue-specific Cre-ER activity independent of exogenous tamoxifen administration, which is most pronounced in the pancreas and the liver. This may result from a number of factors including tissue-specific basal expression from the Col1a1 locus, tissue-specific exposure to endogenous estrogens that may activate Cre-ER, and the rate of tissue-specific protein turnover. Tissues that are less affected include the bone marrow and the brain. This may limit the scope of experiments that can be performed with the Col1a1 allele to younger mice, a shorter timeframe for sequential mutagenesis, and/or specific organs especially in older mice. In contrast, we did not observe any leakiness from Cre-ER in the Col1a1 mice. Thus, the age of mice for experiments with this allele does not appear to be a critical experimental variable. In primary sarcomas initiated by AdenoFlpO from Col1a1; Kras; p53; Rosa26 mice, we were able to activate Cre-ER in tumor cells by intratumoral administration of 4hydroxytamoxifen or multiple doses of systemic 4-hydroxytamoxifen. However, experiments with intraperitoneal injection of a single dose of tamoxifen failed to activate CreER in primary sarcomas when they were approximately 200mm. Therefore, the delivery of an adequate level of active tamoxifen metabolites into the tumor will be necessary to apply this system in established sarcomas. In addition to injecting 4-hydroxytamoxifen into the tumor or systemically, another approach that could be utilized is delivery of tamoxifen in the food (Schonhuber et al., 2014). Recently, in vivo manipulations of the mouse genome have been achieved using the CRISPR/Cas9 system to generate tumors such as those in the liver and lung via hydrodynamic injections (Xue et al., 2014), adenoviral delivery of both sgRNA and Cas9 D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t (Sanchez-Rivera et al., 2014), or adenoviral delivery of sgRNA and Cre into Rosa26 loxP-Cas9 mice (Platt et al., 2014). There may be distinct advantages and disadvantages to using either the CRISPR/Cas9 or the Cre-loxP recombinase systems to model cancer depending on the application. For example, the CRISPR/Cas9 system does not require generation of mice with multiple mutant alleles. Therefore, the CRISPR/Cas9 system enables testing the impact of different gene mutations on a cancer model more quickly. However, the Cre-loxP recombinase system can be much more efficient in generating a specific gene mutation with higher efficiency. One exciting application of the CRISPR/Cas9 system would be to combine the efficiency of the CRISPR/Cas9 system with the spatial and temporal control of stepwise genomic manipulations with the Flp-FRT mediated Cre-ER mouse strains. Specifically, the Col1a1 mouse strain could be used in combination with Rosa26 loxP-Cas9 mice such that in vivo genome-scale screening would be possible to evaluate cooperating mutations for cancer initiation and maintenance. In summary, we have generated two complementary novel Flp-FRT regulated CreER mouse strains that can be used for sequential mutagenesis with the Flp-FRT and the Cre-loxP systems. The Col1a1 allele can be combined with FlpO, FRTflanked, and loxP regulated genes to generate mice where distinct mutations can be expressed in neighboring cells to study intercellular communications such as those between tumor parenchymal and stromal cells. Furthermore, the Col1a1 allele can be combined with FlpO, FRT-flanked, and loxP regulated genes to FRT generate mice in which two distinct mutations can be expressed in a single cell sequentially. We anticipate that this allele will provide a tightly regulated tool to study sequential mutagenesis in cancer development and to test the role of genes in tumor maintenance by mimicking therapeutic target inhibition or activation during cancer therapy. D ise as e M od el s & M ec ha ni sm s D M M Ac ce pt ed m an us cr ip t MATERIALS AND METHODS All animal experiments were performed according to protocols approved by the Duke University Institutional Animal Care and Use Committee. Generation of novel mouse models Knock-in alleles Col1a1 Cre-ER-T2-FRT and Col1a1 Cre-ER-T2 were generated by targeting the FRT-Cre-ER-FRT-NEO or the FRT-STOP-FRT-Cre-ER-NEO constructs to the Col1a1 locus in 129/SVJae embryonic stem cells (ES cells). 129/SVJae ES cells were selected in neomycin (G418), and screened for homologous recombination at the Col1a1 locus by PCR and Southern Blot. ES cells that were successfully targeted at the Col1a1 locus were injected into C57BL/6 blastocysts, which were then implanted into pseudo-pregnant mice. Male chimeras were bred to female C57BL/6 mice and the resulting progeny with brown colored coat (i.e. 129/SVJae-derived) were analyzed for germline transmission of the targeted alleles. Mice with germline transmission were crossed to the Rosa26 (Raymond and Soriano, 2007), which removed the attB/attP-flanked neomycin