SBIR-STTR Award

This project aims to rapidly produce rat mutants through a transposon-based method of mutagenesis. The rat is a favored model for many types of human disease for which mice are not suitable. The rat is the most relevant model for cardiovascular and hypertension disease, with a heart rate much more similar to that of humans compared to mice. As opposed to the mouse, rats and humans also share a more similar number of cytochrome P450 genes, perhaps explaining why the rat has been 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. Unlike the acrocentric mouse genome, the rat and human genomes are predominantly metacentric, making chromosomal comparisons more relevant for modeling human genetic diseases. However, rat mutants cannot be easily generated through traditional methods of homologous recombination in embryonic stem (ES) cells, because rat ES cells cannot be cultured or manipulated. Alternative methods are inefficient and costly. While the rat has not had the ease of genetic manipulation that the mouse has had over the past twenty years, it certainly possesses physiological, anatomical, and chromosomal similarities to humans that make for a more relevant model of human disease. Now, this proposal seeks to rapidly produce rat mutants through the development of hyperactive transposons for germ line insertional mutagenesis. This approach uses randomly integrating transposons, which enables the rapid identification of sequence-tagged mutation sites. The Aims will focus on synthesizing hyperactive transposases from three different families of transposons, each with unique insertion characteristics. The current standard for mutagenesis in rodents using DNA transposons is a rate of 1-3 insertions per gamete in rats and mice. However, to be commercially viable and produce at least one null allele in each offspring, this transposition rate must be substantially improved. Our goal is to increase the mutation frequency to a level at least 25X greater than the transposons currently used for rodent mutagenesis. Hyperactive transposases will be the direct product from this study, while the ultimate Phase II goal will be the generation of rat mutants (MutaRats) for modeling human disease. Offspring of MutaRats (MutaRat Knockout Rat Models) will not be phenotyped or mated to produce a breeding colony, but sperm will be isolated from mutant males and cryogenically frozen for future retrieval and fertilization using already existing technology. Mutant animals 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.

Public Health Relevance:
About 89% of all compounds tested for drug use fail during the final stages of approval due to unacceptable side effects or a lack of efficacy. There is a clear need to screen drugs more effectively in animals during preclinical testing before entering the most expensive phases of drug testing in humans. This project is designed to employ a new method using mobile DNA elements (or "jumping genes") for the rapid and economical production of a large variety of mouse and rat models of human disease, which will enable a greater scrutiny of candidate drugs and will facilitate more favorable testing in humans.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
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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. , ,

Public Health Relevance:
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.

Thesaurus Terms:
Addiction, Drug;Address;Affect;Animal Model;Animal Models And Related Studies;Animals;Arthritis;Backcrossings;Behavior Disorders;Behavioral;Biomedical Research;Blastocyst;Blastocyst Structure;Blastosphere;Blood Vessels;Blotting, Southern;Body Tissues;Brain Imaging;Cardiac;Cardiovascular;Cardiovascular Body System;Cardiovascular System;Cardiovascular System (All Sites);Cataloging;Catalogs;Cell Count;Cell Culture Techniques;Cell Differentiation;Cell Differentiation Process;Cell Isolation;Cell Line;Cell Lineage;Cell Lines, Strains;Cell Number;Cell Segregation;Cell Separation;Cell Separation Technology;Cellline;Cells;Characteristics;Chemical Dependence;Code;Coding System;Common Rat Strains;Communities;Complement;Complement Proteins;Cryofixation;Cryopreservation;Cytochrome P-450;Cytochrome P-450 Enzyme System;Cytochrome P450;Dna;Dna Sequence;Dna Transposons;Dna Analysis;Dna Blotting;Data;Defect;Deoxyribonucleic Acid;Dependence, Drug;Derivation;Derivation Procedure;Diabetes Mellitus;Disease;Disorder;Drug Addiction;Drug Dependency;Elig;Es Cell;Elements;Eligibility;Eligibility Determination;Embryo;Embryo, Preimplantation;Embryonic;Endothelial Cells;Engineering;Engineerings;Enhancers;Event;Exhibits;Expression Profiling;Expression Signature;Family;Funding;Gene Expression;Generations;Genes;Genetic Alteration;Genetic Change;Genetic Defect;Genetics-Mutagenesis;Genomics;Germ Lines;Goals;Heart;High Throughput Assay;Hour;Housekeeping Gene;Human;Human, General;In Situ;In Vitro;Individual;Knock-Out;Knockout;Lead;Libraries;Mammals, Mice;Mammals, Rats;Mammals, Rodents;Man (Taxonomy);Man, Modern;Marketing;Mediating;Mice;Modeling;Molecular Biology, Mutagenesis;Molecular Fingerprinting;Molecular Profiling;Mother Cells;Murine;Mus;Mutagenesis;Mutate;Mutation;Nih;National Institutes Of Health;National Institutes Of Health (U.S.);Nature;Nerve Cells;Nerve Unit;Nervous;Neural Cell;Neurocyte;Neurons;Noise;Organ System, Cardiovascular;P450;Pb Element;Pharmaceutical Agent;Pharmaceuticals;Pharmacologic Substance;Pharmacological Substance;Pharmacology And Toxicology;Phase;Phenotype;Plant Embryos;Plasmids;Policies;Population;Probability;Production;Progenitor Cells;Protocol;Protocol Screening;Protocols Documentation;Public Health;Publishing;Quality Control;Rat;Rattus;Reporter;Reporter Genes;Reporting;Research;Research Resources;Resources;Rodent;Rodentia;Rodentias;Screening Procedure;Seeds;Site;Sleeping Beauty;Sorting - Cell Movement;Southern Blotting;Sperm;Spermatozoa;Stem Cells;System;System, Loinc Axis 4;Technology;Therapeutic Human Experimentation;Therapeutic Research;Tissues;Toxicology;Transfection;Transposase;Transposons, Dna;United States National Institutes Of Health;Vascular, Heart;Zygotes, Plant;Arthritic;Behavioral Disorder;Blastocyst;Blastula;Brain Visualization;Cell Bank;Cell Sorting;Circulatory System;Cold Preservation;Cold Storage;Controlled Release;Cultured Cell Line;Diabetes;Disease/Disorder;Drug Discovery;Embryonic Stem Cell;Experiment;Experimental Research;Experimental Study;Falls;Genome Mutation;Heavy Metal Pb;Heavy Metal Lead;High Throughput Analysis;High Throughput Screening;Human Disease;In Vivo;Insertion Sequence;Insight;Interest;Male;Mobile Dna;Model Organism;Molecuar Profile;Molecular Signature;Mutant;Neural;Neuronal;Novel;Phase 1 Study;Phase 2 Study;Public Health Medicine (Field);Public Health Relevance;Rat Genome;Relating To Nervous System;Research Study;Screening;Screenings;Seed;Sorting;Sperm Cell;Stem;Stem Cell Differentiation;Stem Cell Of Embryonic Origin;Stem Cell Technology;Success;Tool;Transposon Element;Transposon/Insertion Element;Vascular;Vector;Zoosperm