The rat is a favored model for many types of human disease for which mice are not suitable. As opposed to the mouse, rats and humans also share more similarity in their cytochrome P450 genes, making the rat a more useful model for toxicology and pharmacology studies. The rat is also a favored model for diabetes, arthritis, behavioral disorders (including drug addiction), and brain imaging. However, until recently, generating engineered mutations has been problematic due to the lack of rat stem cell lines capable of contributing to the germ line, and the lack of efficient technologies to modify genomic sequences. Transposagen Biopharmaceuticals has pioneered the use of mobile DNA elements (e.g., transposons) to generate insertional mutations in the rat germ line. To date, we have over made over 100 insertional mutant lines (referred to as TKOTM Knockout Rat Models). This approach utilizes gene-trap strategies to select for randomly integrated transposons, which enable the rapid identification of sequence-tagged mutation sites. In Phase I studies, we focused on synthesizing hyperactive transposases, from three different families of transposons, Sleeping Beauty (SB), piggyBac (PB), and TcBuster (TcB), to increase the efficiency of transposition in the germ line. We report the successful generation of a number of hyperactive transposases in the PB family. In Phase II studies, we will generate a rat embryonic stem (rES) cell bank containing over 200,000 dual reporter gene-trap insertional mutations;a EGFP reporter system will be used as a polyA trap to maximize the probability of generating insertional mutations in each of the approximately 30,000 rat genes, and a promoterless tdTomato reporter to screen for lineage-specific gene disruptions. In the long term, we intend to use the rES cell bank to generate rat knockout lines in each locus. In Phase II studies, we will focus on developing transposon-mediated knockout lines in neural, cardiac, and endothelial cell lineages, using the hyperactive PB transposases created in Phase I studies, with the aim of generating a bank of knockout lines that that will be valuable for a wide variety of applications such as toxicology, behavioral, and cardiovascular research. We will develop high-throughput in vitro differentiation protocols to screen pools of rES cells cultured in 96-well formats. Potential mutations in neural, cardiac, and endothelial cell lineages will be identified by screening for wells that contain tdTomato positive cells after lineage-specific differentiation protocols. rES cell pools in positive wells will be subcloned and re-screened to identify the individual clone that carries the potential lineage specific mutation. Genomic DNA will be isolated and used as template for splinkerette PCR, which is used routinely to amplify sequences that flank DNA insertions. We will determine the genomic sites for each insertion and screen each gene for lineage-specific expression to rule out insertions that affect ubiquitously expressed loci. We will develop two products for academic and pharmaceutical end users. First, we will generate chimeric animals from selected rES cell clones by injecting these cells into host blastocysts, and segregate away the """"""""irrelevant"""""""" mutations by several rounds of backcrosses to generate additional TKOTM Knockout Rat Models for human diseases. Second, we will market rES cell clones, containing mutations in genes that are beyond our core interests to academic and pharmaceutical end users. Mutant animals, sperm isolated from mutant males, and mutant rES cells will be distributed by the National Rat Resource and Research Center and shared with the academic community according to NIH policies for sharing model organisms for biomedical research.
In the application """"""""Creation of hyperactive transposases for mutagenesis in rodents,"""""""" we are seeking Phase II funding to use the novel transposases we created to generate new models of human disease. We have demonstrated the value of creating transposon-mediated mutations to model human diseases for basic and therapeutic research application. All of our previous models were obtained by random mutagenesis. In this proposal we outline studies that will enable us to identify transposon mediated mutations that will likely affect neural, heart, or blood vessel function. Such rat models will provide new and valuable tools to develop new therapies in classes of diseases that are particularly prevalent in humans. Thus, if successful, this project would benefit many goals of public health by making the production of mutations in the rat that model human diseases readily accessible to the research community.