Development of Cre driver rat strains for creating conditional and physiologically relevant models of human diseases

The goal of this proposal is to establish a Cre loxP rat resource for conditional and physiologically predictive rat models of human diseasesThe laboratory ratRnorvegicusis a central experimental animal in several fields of biomedical researchsuch as cardiovascular diseasesaginginfectious diseasesautoimmunitycancer modelstransplantation biologyinflammationcancer risk assessmentindustrial toxicologypharmacologybehavioral and addiction studiesand neurobiologyUp till recentlythe ability of creating genetically modified rats has been limited compared to that in the mouse mainly due to lack of genetic manipulation tools and technologies in the ratRecent advances in nucleasessuch as CRISPR Casclustered regularly interspaced short palindromic repeats CRISPR associated proteinhave been successfully used to constructknockoutrat models by injecting gene targeting molecular complexes directly into an embryo for offspring productionavoiding the need to use any type of stem cellsHoweverCRISPR of its current state works inefficiently for gene knockin in mice or ratslimiting its application in generating site specific transgenic rat modelsZinc finger nucleaseZFNhas been successfully used to generate Cre knockin rat modelsbut it requires expertise in ZFN design and validationand is also costlyWith a phase I SBIR awardgrantRGMAwe successfully developed the rat TARGATTTM system that enables fastefficient and site specific introduction of exogenous genetic elements into the rat genomeWe generated rat models with adocking siteattPinserted specifically at a transcriptionally activesafe genomic locus named rHUsing the bacteriophage integrase technologytrademarked asTARGATTTMthese attP containing rats can be used as embryo donors for pronuclear microinjection to insert any gene of interest at the attP siteIntegrases such as phiCor Bxbcarries out efficientunidirectional recombination between two non identical sitesattP and attBUpon microinjection of an attB transgene plasmidtogether with integraserecombination between attP and attB results in an insertion of the transgene precisely at the attP site in the rat genomeThis TARGATTTM technology allows a fastefficient generation of knockin rat models containing any gene of interest with consistentstableand guaranteed gene expressionThe current Phase II proposal builds logically on the result of our Phase I study and aims to use the TARGATTTM technology to build a collection of site specific transgenic rats as tool modelsMore specificallywe aim to generate Cre driver rat models that will allow controlled gene expression or knockoutconditional modelsboth temporally and spatially through the Cre loxP systemHundreds of Cre driver mouse models and overCre mice distributed by the Jackson Laboratory alone have proven to be crucial in building physiologically predicative mouse models of human diseasesIn comparisonthere are less than a dozen of Cre rat models reportedindicating an urgent need in establishing multiple Cre rat lines to ensure desired rat models can be generatedWe propose to generateas a starting pointa total ofCre rat lines and one Cre reporter test line in this studyincluding eighteen Cre lines on neural specific and two lines on cardiovascular specific Cre expressionAdvantages of using our TARGATTTM integrase technology areTransgene integration happens at a pre selected and transcriptionally active locus to ensure that Cre is expressedSite specific knockin rat models are made by direct injection of DNAmRNAor protein into the rat zygotesbypassing rat ES cellsGene integration efficiency is higher than CRISPR and cost is lower than ZFNSuccessful execution of this project will create a novel and much needed resource for the bio medical community who employ rat models for their studies of human diseasesOur long term goal is to further expand the resource to include more Cre rats and rats with loxP flankedfloxedalleles for conditional rat model generation! !