Development of novel rat models for site-specific transgene integration

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The goal of this proposal is to develop novel rat strains that can be used to efficiently generate site-specific transgenic rat models via pronuclear microinjection. 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. The isolation and establishment of rat embryonic stem (rES) cells by Drs. Ying and Smith's groups at the end of 2008 revolutionized the capability of making genetically modified rat models. As a result, rat transgenics is becoming as precise and powerful as has been the case in mice, yielding better models of human diseases. Recent advances in nucleases such as Zinc-finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs) have been successfully used to construct 'knockout' rat models by injecting gene targeting molecular complexes directly into an embryo for chimeric offspring production, avoiding the need to use any type of stem cells. To facilitate the generation and use of rat models of human diseases, it is critical to develop systems that enable fast, efficient and precise introduction of exogenous genetic elements into the rat genome. Here, we propose to use the bacteriophage integrase system to direct transegene integration at transcriptionally active genomic loci with higher integration efficiency. Integrases such as phiC31 or Bxb1 carries out efficient, unidirectional recombination between two non-identical sites, attP and attB. In this proposal, we will identify transcriptionally active loci in the rat genome and insert attP sites at such loci using TALEN-mediated homologous recombination (HR). These attP-containing rats will be used as embryo donors for pronuclear injection of the transgene on an attB plasmid. In the presence of integrases, recombination between attP and attB results in an insertion of the transgene precisely at the attP site in the rat genome. This technology will allow a fast, efficient generation of knockin rat models containing any gene of interest with consistent, stable, guaranteed gene expression. Advantages of this integrase-based technology are: (1) Transgene integration happens at pre-selected and transcriptionally active loci; (2) Site-specific knockin rat models are made by direct injection of the DNA into the rat zygotes, bypassing rES cells; (3) Gene integration efficiency is much higher, but off-target events are lower compared to ZFNs or TALENs. Successful execution of this project will create a cost-effective method and valuable resources for the bio-medical community who employ rat models for their studies of human diseases. ! !