The NIMH transgenic core facility has several major functions: 1) to produce transgenic research animals for neuroscience research, 2) to support research with associated techniques in genetic research in neuroscience, 3) to develop new transgenic techniques and model systems and 4) to engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year include: a) 24 transgenic mouse projects produced by oocyte injection of DNA or CRISPR constructs, with multiple lines produced for each project. b) 5 transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. 2) Technical Support a) 63 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 44 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) 19 marmosets have been born. In the last year it has been critical to increase the size of the colony as new research is proposed in the institute. d) Transgenic project design and assistance have continued to be significant to NIH neuroscience labs that lack experience in producing transgenic animals. 3) Technical development a) Nuclease-mediated genetic engineering: There are unique features of a mouses embryonic development that make it possible to manipulate its genome. Mouse embryonic stem cells (ESC) dissected from early embryos can be grown in a dish, and still maintain the ability to generate a complete mouse from a single cell. The genes of that single cell can be altered and then expanded and transferred into a foster mother. Other species, including primates, have been refractory to the same technique that work so well for mice. The result has been an ever expanding collection of transgenic mouse lines that scientists use to test the role of virtually every one of the roughly 20,000 genes that make up a mouse genome. The CRISPR/cas9 system can be used to target genes more efficiently and as specifically as in mouse ESC, but is applicable to other species. The core has been focusing on using CRISPR/cas9 to generate conditional knock out genes. These are genes in which recombination sites are innocuously inserted to flank critical regions of a gene. Only when a recombinase is expressed in the celldelivered from another transgene or a transient virusis the gene knocked out. Subtle changes in the gene can also be made, but making a conditional modification requires the replacement of large chunks of a gene. Initially, the core worked with the NIDA virus production core to make rat transgenic lines with both standard and CRISPR constructs. Moreover, since the beginning of 2016 the core has collaborated with Nick Ryba in the National Institute of Dental and Craniofacial Research (NIDCR) to improve the efficiency of modifying genes by replacing large segments. We have optimized concentration of the injected material, the use of reagents to optimize homologous recombination of donor sequences, the site of injection in embryos, and the construction of nucleic acid targeting vectors. These techniques are now being applied to make mouse and rat transgenics, with the ultimate aim of making the technique efficient enough to use in species with smaller numbers of offspring, and longer gestation and maturation times, such as marmosets. b) Transgenic marmosets: In collaboration with Erika Sasaki at the Central Institute for Experimental Animals in Kawasaki, Japan the core has produced a transgenic marmoset that expresses a genetically encoded calcium indicator (GECI) and Kusabira Orange, a fluorescent marker protein. This transgenic monkeys tissues glow orange when they are excited with a specific wavelength of green light. To expand a line of animals that express this gene we have developed an artificial insemination procedure that has resulted in multiple pregnancies in the NIMH colony and in the Xiaoqin Wangs colony at Johns Hopkins University. c) Transgenic rat lines: Rat lines produced in the NIMH Transgenic core have been deposited in the RRRC (Rat Resource and Research Center) and are being distributed from that organization. Over ten lines that express CRE recombinase in specific neuronal subtypes were produced in collaboration with the NIDA virus production core, which characterized the pattern of expression. Two lines are being used in studies of neurogenesis. In one approach neural stems cells that express the thymidine kinase (TK) transgene can be specifically ablated. Jason Snyder (U British Columbia) and Heather Cameron (NIMH) Another inbred line that expresses the orange fluorescent protein (OFP) is being used by Alan Korestskys group (NINDS) to study the potential of transplanted neural stem cells. e) Transgenic voles Recently, the core initiated a project to make transgenic voles. This project for Scott Young (NIMH) and Adam Smith (University of Kansas) takes advantage of CRISPR technologies to manipulate the genes of an animal with interesting social behavioral traits. f) Support techniques: several techniques are under development to increase the capacity of the core's support functions. Freezing mouse sperm and improving IVF by using newer methods is a major effort. Freezing rat sperm and completing IVF in rats at an acceptable level is a challenging task in all laboratories.
Showing the most recent 10 out of 37 publications
