Dual Recombinase Research Articles

The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis.

ABSTRACTNovel 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 cell 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-ERT2 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-ERT2 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-ERT2 alleles into the endogenous Col1a1 locus for ubiquitous expression. In the Col1a1FRT-Cre-ER-T2-FRT mice, Flp deletes Cre-ERT2, so that Cre-ERT2 is only expressed in cells that have never expressed Flp. In contrast, in the Col1a1FRT-STOP-FRT-Cre-ER-T2 mice, Flp removes the STOP cassette to allow Cre-ERT2 expression so that Cre-ERT2 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.

Abstract LB-167: Tumor cells, but not endothelial cells, mediate the eradication of primary cancers by radiation therapy

Abstract Radiation therapy is frequently utilized in the clinic as curative treatment for cancers, but the contribution of endothelial cells to tumor response to radiation remains controversial. Using the two highly efficient site-specific recombinases, Cre and FlpO, we have generated primary tumors in mice with different gene mutations specifically in tumor cells and stromal cells. With this dual recombinase technology, we selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. We found that deletion of Bax in endothelial cells did not affect tumor response to radiation, but Atm deletion increased radiation-induced death of tumor endothelial cells and prolonged tumor growth delay following a non-curative dose of radiation. However, following a curative dose of radiation, Atm deletion in endothelial cells did not affect growth delay of primary tumors and failed to increase local control. In contrast, deletion of Atm in tumor cells increased local control of primary tumors by radiation therapy. These results demonstrate that tumor cells rather than endothelial cells are the critical targets that regulate tumor eradication by radiation therapy. They also emphasize the importance of using primary models of cancer to study the role of stromal cells in tumor development and response to therapy. Citation Format: Everett J. Moding, Chang-Lung Lee, Katherine D. Castle, Patrick Oh, David G. Kirsch. Tumor cells, but not endothelial cells, mediate the eradication of primary cancers by radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-167. doi:10.1158/1538-7445.AM2014-LB-167

Dual Recombinase Technology Defines a Therapeutic Window for ATM Inhibitors to Selectively Radiosensitize Tumors

Dual Recombinase Technology Defines a Therapeutic Window for ATM Inhibitors to Selectively Radiosensitize Tumors

Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.

Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.

A FRT-flankedp53mouse to generate primary tumors with Flp recombinase

SUMMARYThe site-specific recombinases Cre and Flp can mutate genes in a spatially and temporally restricted manner in mice. Conditional recombination of the tumor suppressor gene p53 using the Cre-loxP system has led to the development of multiple genetically engineered mouse models of human cancer. However, the use of Cre recombinase to initiate tumors in mouse models limits the utilization of Cre to genetically modify other genes in tumor stromal cells in these models. To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2–6 of the endogenous p53 gene in mice to generate a p53FRT allele that can be deleted by Flp recombinase. We show that FlpO-mediated deletion of p53 in mouse embryonic fibroblasts impairs the p53-dependent response to genotoxic stress in vitro. In addition, using FSF-KrasG12D/+; p53FRT/FRT mice, we demonstrate that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. p53FRT mice will enable dual recombinase technology to study cancer biology because Cre is available to modify genes specifically in stromal cells to investigate their role in tumor development, progression and response to therapy.

Engineering Cell-Permeant FLP Recombinase for Tightly Controlled Inducible and Reversible Overexpression in Embryonic Stem Cells

Combined application of DNA recombinases Cre and FLP enables tightly controlled independent and/or sequential gene regulations. However, in practice, such dual recombinase strategies are hampered by the comparably low efficiency of the FLP recombinase. Here, we present the engineering of a recombinant cell-permeant FLP protein (TAT-FLP) that induces recombination in >75% of fibroblasts and mouse as well as human embryonic stem (ES) cells. We show that TAT-FLP ideally complements the strength of cell-permeant Cre recombinase for genetic engineering as exemplified by FLP-ON-Cre-OFF, an inducible transgene expression cassette that enables tightly controlled expression in a reversible manner. We exemplify this concept by conditional overexpression of LacZ and the caudal-related homeobox transcription factor CDX2. We expect our FLP transduction system to become widely useful for numerous genetic interventions addressing complex biological questions and the generation of transgene-free therapeutically applicable ES cell-derived cells.

Flp recombinase regulated lacZ expression at the ROSA26 locus.

Manipulation of gene expression patterns through gene targeting in mouse embryonic stem (ES) cells (Thomas and Capecchi, 1987) and the more recent advent of conditional mutagenesis has revolutionized mouse genetics. The recombinase system most commonly used for conditional alleles in mouse is Cre/loxP derived from bacteriophage P1 (Sauer and Henderson, 1988). While less utilized, FLP/FRT derived from S. cerevisiae (O’Gorman et al., 1991) has been elegantly used by Martin and colleagues in combination with Cre to create a series of alleles for Fgf8 of varying strength (Meyers et al., 1998). The utility of Flp as a robust tool for mammalian genetics has been markedly improved by Stewart and colleagues who cleverly used cycling mutagenesis to select for variant alleles of Flp that carry out recombination at high efficiency in mammalian cells (Buchholz et al., 1998). Experimental strategies to sequentially inactivate and reactivate a given gene, or to test epistatic relationships between genes in somatic cells, are now possible using a combination of Cre with the enhanced Flpe. As Cre and Flp transgenes used to regulate conditional alleles may not recapitulate the expected pattern of expression (e.g., Tallquist and Soriano, 2000), it is essential to characterize the activity of these recombinase alleles to correctly interpret the phenotypes of conditionally mutant animals. Whereas several reporter lines for Cre have been described, we have found the allele created by Soriano in which Cre regulates lacZ expression at the ROSA26 locus to be especially useful (Soriano, 1999). ROSA26 insertions are ubiquitously expressed and show no overt phenotype when homozygous (Zambrowicz et al., 1997). We therefore created an analogous ROSA26 allele, which we term R26FR, to monitor Flp activity in vivo. The targeting vector contains an FRT-flanked neomycin resistance gene with four transcriptional stop signals placed upstream of a -galactosidase-neo fusion ( -geo; Friedrich and Soriano, 1991) all inserted into ROSA26 genomic sequence (Fig. 1a). The vector was transfected into V6.5 ES cells (Eggan et al., 2001), 48 clones were analyzed by Southern blotting, and 24 correctly targeted clones were identified (Fig. 1b). To demonstrate that the R26FR clones could activate – geo, a CMV-FLPe expression plasmid (Buchholz et al., 1998) was introduced by transient lipofection. One hundred ninety-two clones were picked and aliquots analyzed for – galactosidase activity by X– gal staining. Both Flpe-recombined (Fig. 2a) and parental clones (Fig. 2d) were identified, and absence or presence of the stop cassette was confirmed by Southern blotting (Fig. 1b). To create the reporter mouse strain, completely ESderived mice (Eggan et al., 2001; Nagy et al., 1990) were made by injecting targeted clones into tetraploid blastocysts. Four viable pups were produced, and two were used to derive tail fibroblast cell lines. To demonstrate functionality of the reporter in somatic cells, Flpe was cloned into an MSCV-IRES-GFP retroviral vector (Van Parijs et al., 1999) and used to infect the transformed reporter cell lines. Cells sorted for and against GFP expression were enriched by FACS and compared with those infected with an antisense Flpe retroviral control vector. Sorted cells were stained with X-gal and analyzed by Southern blotting, which demonstrated conditional recombination (Fig. 1b) and -galactosidase activity (Fig. 2b) dependent upon Flpe. To confirm activity of the reporter allele in vivo, R26FR mice were crossed to a -actin-Flpe transgenic mouse (Rodriguez et al., 2000). Embryos dissected at day 10.5 pc were stained with X-gal and showed ubiquitous Flp-mediated activation (Fig. 2c). The purpose of this FLP reporter allele is to specify the extent of FLP activity with a convenient and widely used enzymatic activity, -galactosidase. Development of FLP/ FRT tools such as our R26FR reporter allele and the recently described alkaline phosphatase reporter (Awatramani et al., 2001) improve the ability to characterize Flpe alleles in vivo and therefore expand the opportunities for using precisely regulated dual recombinase experimental strategies.

A recombinase-mediated transcriptional induction system in transgenic plants.

We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed. The system developed here uses a heat-shock-inducible Cre to excise a DNA fragment flanked by lox sites, thereby generating a constitutive GUS reporter gene under control of the CaMV 35S promoter. Heat-shock-mediated excision of several, independent lines resulted in varying degrees of recombination-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over-expression, or under-expression by antisense, would otherwise affect embryo, seed or seedling viability.