Transgenic Resources for Neuroscience Research

NIMH转基因核心设施有几个主要功能：1)为神经科学研究生产转基因研究动物，2)开发新的转基因技术和模型系统，3)参与合作项目，促进神经科学研究的遗传方法。

The NIMH transgenic core facility has several major functions: 1) to produce transgenic research animals for neuroscience research, 2) to develop new transgenic techniques and model systems, and 3) to engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year include projects: a) 12 transgenic rodent projects produced by CRISPR constructs, with multiple lines produced for each project in rats or mice. b) no projects that use standard plasmid constructs, and c) one project developing three, genetically unique embryonic stem cell lines. 2) Technical Support a) 56 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 25 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) 2 projects were completed using unique surgical techniques. 3) Technical development a) In this year, during the Coronavirus Disease (COVID-19) pandemic, as most researchers worked away from their usual laboratories and animal colonies it became critical to preserve valuable research animal models. Without a predictable time when researchers could return to the physical workplace, there was the danger of losing lines that had taken extensive resources to develop. For that reason, the core facility worked through this period in order to first preserve lines by cryopreservation and later to recover lines for renewed research. Fifty-six lines were cryopreserved. Before the end of the fiscal year, at least 10 lines will be rederived and ready for experiments that will follow when research begins anew. b) Over the last year the core has generated most transgenic rodents for neuroscience research using CRISPR technology. Some transgenics have been generated using optimized CRISPR guides and homology arms to insert promoters and coding sequence into transcriptionally neutral genomic sites, such as the ROSA locus of mice and rats. Other transgenics have been produced by knocking in large regions of a gene to add recombination signals flanking coding exons to generate conditional knockouts. Another set of transgenics add CRE or FLP recombinases to existing transcripts in order to maintain the fidelity of expression from endogenous transcriptional promoters. Only rarely does the core produce transgenics using plasmid- or ESC (embryonic stem cell)-mediated transgenic methods. c) The process of generating transgenic marmosets by manipulating embryos to produce a line of germline transgenic animals is no longer a feasible option for most research. These methods are useful for producing lines of transgenics that express recombinases. However, a more effective method of introducing a transgene using AAV vectors that crosses the blood brain barrier and expresses a transgene in neurons in the brain has been developed. In collaboration with the Gradinaru laboratory at Cal Tech, we have produced several animals that carry transgenes and express them in the brain. Further work is being completed to use this technology in the intramural program. d) The ACE2 protein is expressed on the surface of cells and is targeted by the receptor binding domain (RBD) of the SARS-CoV2 virus. This allows the virus to enter the cell and create a productive infection. Research animals have a range of susceptibility to this virus. Modifying an animals ACE2 gene to match the human gene may advance the study of COVID-19, especially the neurological features that are poorly understood. This modified gene has been introduced in culture systems (cells and animal zygotes) to test this method. e) A panel of rats that express the CRE recombinase was generated in a collaboration with investigators at NIDA. Those lines continue to be distributed through the Rat Resource and Research Center. Other CRE-expressing lines will be distributed through RRRC once they are characterized. f) Mouse ESC (embryonic stem cell) lines that express the green fluorescent protein continue to be licensed.