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 114 projects: a) 19 transgenic rodent projects produced by CRISPR constructs, with multiple lines produced for each project in rats or mice. b) 2 projects that use standard plasmid constructs, and no projects using embryonic stem cell techniques. 2) Technical Support a) 63 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 27 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) 3 projects were completed using unique surgical techniques. 3) Technical development a) 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. b) 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. c) 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. d) Mouse ESC (embryonic stem cell) lines that express the green fluorescent protein continue to be licensed. Representative publications that resulted from NIMH Transgenic core resources during the report period: Weeden, CSS, Mercurio, JC, Cameron HA. A role for hippocampal adult neurogenesis in shifting attention toward novel stimuli. Behav Brain Res 2019. August 13:112152 doi: 10.1016/bbr.2019.112152 PMID: 31419520 Schoenfeld TJ, Rhee D, Martin L, Smith JA, Sonti AN, Padmanaban V, Cameron H. New neurons restore structural and behavioral abnormalities in a rat model of PTSD. Hippocampus 2019 Sep 29:29(9):848-861. Doi:10:1002/hipo.23087.PMID 30865372 Kavarthapu R, Anbazhagan R, Raju M, Morris CT, Pickel J, Dufau ML. Targeted knock-in mice with human mutations in GRTH/DDX25 reveals the essential role of phosphorylated GRTH in spermatid development during spermatogenesis. Hum Mol Genet. 2019 Apr 22. pii: ddz079. doi: 10.1093/hmg/ddz079. Epub ahead of print PMID: 31009948 Bck S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope BT, Hamra FK, Richie CT, Harvey BK. Neuron-specific genome modification in the adult rat brain using CRISPR/cas9 transgenic rats. Neuron. 2019 Apr 3;102(1):105-119.e8. doi: 10.1016/j.neuron.2019.01.035. Epub 2019 Feb 18. PMID: 30792150 Pardo-Garcia TR, Garcia-Keller C, Penaloza T, Richie CT, Pickel J, Hope BT, Harvey BK, Kalivas PW, Heinsbroek JA. Ventral Pallidum is the primary target for accumens D1 projections driving cocaine seeking. J Neurosci. 2019 Mar 13;39(11):2041-2051. doi: 10.1523/JNEUROSCI.2822-18.2018. Epub 2019 Jan 8. PMID: 30622165 Zallar LJ, Beurmann S, Tunstall BJ, Fraser CM, Koob GF, Vendruscolo LF, Leggio L. Ghrelin receptor deletion reduces binge-like alcohol drinking in rats. J Neuroendocrinology 2019 Jul;31(7): 20; doi: 10.1111/jne.12663 PMID: 30456835 Zallar LJ, Tunstal BJ, Richie CT, Zhang YJ, You ZB, Gardner EL, Heilig M, Pickel J, Koob GF, Vendurscolo LF, Harvey BK, Leggio L. Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout Wistar rat model. Int J Obes(London) 2019 Feb:43(2):344-354 doi: 10.1038/S41366-018-0013-5 PMID:29453460
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