The goal of this proposal is to establish a Cre/loxP rat resource for conditional and physiologically predictive rat models of human diseases. The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Up till recently, the 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 rat. Recent advances in nucleases, such as CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9), have been successfully used to construct ?knockout? rat models by injecting gene targeting molecular complexes directly into an embryo for offspring production, avoiding the need to use any type of stem cells. However, CRISPR of its current state works inefficiently for gene knockin in mice or rats, limiting its application in generating site-specific transgenic rat models. Zinc finger nuclease (ZFN) has been successfully used to generate Cre knockin rat models; but it requires expertise in ZFN design and validation, and is also costly. With a phase I SBIR award (grant # 1R43GM108071-01A1), we successfully developed the rat TARGATTTM system that enables fast, efficient and site-specific introduction of exogenous genetic elements into the rat genome. We generated rat models with a ?docking site?, attP, inserted specifically at a transcriptionally active, safe genomic locus named rH11. Using the bacteriophage integrase technology (trademarked as ?TARGATTTM?), these attP-containing rats can be used as embryo donors for pronuclear microinjection to insert any gene of interest at the attP site. Integrases such as phiC31 or Bxb1 carries out efficient, unidirectional recombination between two non-identical sites, attP and attB. Upon microinjection of an attB-transgene plasmid, together with integrase, recombination between attP and attB results in an insertion of the transgene precisely at the attP site in the rat genome. This TARGATTTM technology allows a fast, efficient generation of knockin rat models containing any gene of interest with consistent, stable, and guaranteed gene expression. The 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 models. More specifically, we aim to generate Cre driver rat models that will allow controlled gene expression or knockout (conditional models) both temporally and spatially through the Cre/loxP system. Hundreds of Cre driver mouse models and over 100 Cre mice distributed by the Jackson Laboratory alone have proven to be crucial in building physiologically predicative mouse models of human diseases. In comparison, there are less than a dozen of Cre rat models reported, indicating an urgent need in establishing multiple Cre rat lines to ensure desired rat models can be generated. We propose to generate, as a starting point, a total of 20 Cre rat lines and one Cre reporter/test line in this study, including eighteen Cre lines on neural specific and two lines on cardiovascular specific Cre expression. Advantages of using our TARGATTTM integrase technology are: (1) Transgene integration happens at a pre-selected and transcriptionally active locus to ensure that Cre is expressed; (2) Site-specific knockin rat models are made by direct injection of DNA, mRNA, or protein into the rat zygotes, bypassing rat ES cells; (3) Gene integration efficiency is higher than CRISPR and cost is lower than ZFN. Successful 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 diseases. Our long-term goal is to further expand the resource to include more Cre rats and rats with loxP-flanked (floxed) alleles for conditional rat model generation. ! !
NIH ExPorter
NIH ExPorter