Genetically engineered mouse models (GEMMs) have greatly expanded our knowledge of human cancer and serve as a critical tool to identify and evaluate new treatment strategies. However, the cost and time required to generate conventional cancer GEMMs limits their use for investigating novel genetic interactions in tumor development and maintenance. To expand and expedite gene function studies in mice, we have developed a conceptually new approach, referred to as “ESC-GEMM” models (1). This methodology is based on the derivation of mouse embryonic stem cells (ESCs) carrying conditional disease-associated alleles that, in combination, generate a functional model of disease. Additionally, we have layered into these models inducible shRNA technology optimized in our laboratory over the last several years as well as powerful new genome-editing techniques to facilitate gene disruption at different stages of disease. By integrating these technologies (see Figure 5), ESC-GEMMs provide a platform to evaluate the contribution of a broad range of candidate “disease genes” in parallel or to develop large cohorts of tailored experimental animals for pre-clinical treatment studies without further intercrossing of engineered mouse strains.
Figure 5. ESC-GEMM method speeds production of complex mouse models — A) ESC-GEMM approach combines technology from different areas to produce fast and scalable mouse models of disease. B) Schematic representation of approach to generate multi-allelic ES cells carrying defined disease-sensitizing alleles. Additional genetic complexity (via shRNAs / genome editing) can be introduced during ESC culture. C) Comparison of the time for generation of complex mouse models using conventional and ESC-GEMM approach.
The prototypical model consists of ES cells carrying at least four alleles: 1) one or more disease predisposing conditional alleles that can be activated by Cre recombinase (e.g. floxed Apc gene, or a “lox-stop-lox” KrasG12D); 2) a tissue-specific Cre recombinase; 3) a Cre-activatable lox-stop-lox rtTA3 allele to enable tissue specific induction of a tet-responsive transgene; and 4) a “homing cassette” that enables site-specific integration of the tet-responsive transgene (cDNA, shRNA, miRNA) downstream of the col1a1 locus using recombination mediated cassette exchange (RMCE) (6). Other iterations omit the tissue-specific Cre (enabling activation by adenovirus expressing Cre), or link the rtTA allele to a fluorescent reporter (providing lineage tracing of Cre-recombined cells). These ESC- GEMMs can be manipulated in vitro (to introduce additional genetic complexity), for instance, by recombining a genetic element into the homing cassette, and used to produce cohorts of experimental mice by blastocyst injection or tetraploid complementation (Figure 6). Thus, the ESC-GEMM approach enables the production of experimental animals in less than two months. Moreover, as additional genetic alterations are introduced in cell culture, many different genes can be assessed in parallel, without the need for large, expensive breeding colonies.
Using the above principles, we developed a series of ESC-GEMMs to explore the role of tumor suppressor genes in tumor initiation and maintenance, to validate new therapeutic targets, and to explore the on-target toxicities of target inhibition in normal tissues. In one example, we used an ESC-GEMM model of pancreatic cancer to demonstrate that suppression of the PTEN tumor suppressor can accelerate carcinoma formation, and that its re-establishment can lead to tumor regression (2). We are currently producing a range of new models integrating new alleles and genome editing tools into the process. More broadly, we hope through these efforts we can accelerate the study of gene function in vivo for cancer and other diseases.
Figure 6. — ESCs harboring a lox-stop-lox KrasG12D mutant allele, the collagen homing cassette, a Pdx1-cre allele, and a CAGs-lsl-rtTA-IRES Katuska allele were targeted with a GFP-linked shRNA against PTEN. Chimeric mice rapidly develop tumors on dox (to induce the Pten shRNA), and the resulting tumors suppressed PTEN in the epithelial compartment, and expressed the rtTA and shRNA Kate and GFP reporters. Dox withdrawal led to Pten restoration and GFP silencing as expected. This led to dramatic and sustained tumor regression (Saborowski et al., Genes Dev, 2014).