Transgenic Resources for Neuroscience Research

National Institute Of Mental Health

Investigators

Linked publications, trials & patents

Abstract

Summary 1) Production of rodent transgenics Metrics of production over the past year include the following projects: a) 19 transgenic rodent projects produced by CRISPR constructs, with multiple lines produced for each project in rats (4) or mice (15). b) only 4 projects used legacy methods to make transgenic animals with plasmids or embryonic stem cells (ESC). 2) Cryopreservation, rederivation, and transfer of transgenic lines. a) 60 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 28 lines have been rederived, by transferring lines from pathogen-bearing animals into those with defined health status. c) over 2000 lines are maintained as frozen germ cells or embryos; duplicated in two locations, one on campus, one off campus to maintain resilience in emergency situations. 3) Special Concerns during times of limited on-site research In this year as most researchers were separated from their usual laboratories and animal colonies, it became critical to preserve valuable research animal models. Without knowing when researchers could return to the laboratory, there was the danger of losing transgenic animal lines that had taken extensive resources to develop. For that reason, the core facility worked through this period of stasis to first preserve lines by cryopreservation and later to recover lines for renewed research. Sixty lines were cryopreserved. In an ongoing effort, at least 30 lines will be rederived before the end of the fiscal year as researchers have returned to their labs and re-establish their studies with transgenic animals. 4) SARS-Cov2 a) Fibroblast cultures have been propagated using methods developed in the lab (see below) over several years. These cultures have proven useful as in vitro models of ACE2 expression. The ACE2 protein (which acts as a binding site for the SARS-CoV2 virus spike protein) has been introduced into cultured marmoset fibroblasts. Both the marmoset protein and the human protein have been expressed on these fibroblasts to compare the potential for viral infection. Currently, these cells are being immortalized to create reproducible testbeds to evaluate the binding of isolated viral spike protein by other investigators. b) Marmoset embryos grown in culture have been injected with CRISPR constructs that alter the ACE2 gene. Binding of the viral spike protein requires ACE2 protein domains. Two of these domains have been altered in cultured marmoset embryos. The frequency of targeting of the segments of the exons that encode these domains is being optimized. 5) Techniques for transgenic production a) Rodents: Over the last year, most of transgenic rodent lines that have been produced use CRISPR / cas9 nuclease technologies. 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 in order to generate conditional knockouts. Another set of transgenics adds 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 legacy techniques such as plasmid- or ESC (embryonic stem cell)-mediated transgenic methods. b) Marmosets: Manipulating embryos to produce a line of transgenic marmosets is no longer a feasible option for most research. We have developed a more effective method of introducing transgenes into brain cells using AAV vector variants. These unique AAVs can be infused into the peripheral circulation, they avoid accumulating in the liver like most virus, but instead preferentially home to the brain. These viruses cross the blood brain barrier and migrate into the brain. These variants were developed in collaboration with the Gradinaru laboratory at Cal Tech where the variants were selected for our screening in the core. We have produced several animals that carry transgenes and express them in the brain. Further work is being completed to use this technology by intramural as well as neuroscientists around the world. 6) Genetic diversity and reproductive techniques for marmosets Marmosets are endemic to eastern Brazil. Animals that are used for research breed well when housed in groups and furnished with enriched environments. The small numbers of marmosets that were used to initiate research colonies meant that inbreeding has become a problem. In order to increase the diversity of marmoset colonies, the core facility has been leading efforts to find animals that are genetically diverse and use assisted reproductive techniques (ART) to increase diversity in marmoset research colonies. a) fibroblast production for archives and whole genome sequencing: techniques to isolate, purify and expand fibroblasts were optimized for marmoset skin biopsies. Those methods have been posted on the web site of the Marmoset Working Group, and are now being used by other labs including National Primate Centers. b) Fibroblast cryopreservation from all our animals have provided a source of cultured cells that can be used for sequence analysis, induction of pluripotent stem cells or experiments such as those described above. c) Immortalization of these cells, especially when used to express proteins that are expressed and post-translationally modified, provide a system that mimics in vivo function. d) ART methods such as artificial insemination (AI) and in vitro fertilization (IVF) have been optimized and are used routinely in the core. e) Sample preparation for AI or IVF has been used to simplify to promote the exchange of genetic diversity. Sperm samples from males that are genetically distinct can be easily collected and transferred to other colonies where AI or IVF can be used to create hybrid offspring. Samples can retain good progressive motility--the best indication of fertilityover 24 hours for shipping to any location. The cores tripartite mission statement continues to be to 1) deliver transgenic animal models for neuroscience investigators at NIH, this has been accomplished in the last year by as described above. 2) The second directive is to provide other technical services, and in this year it has been the overwhelming role of the core to compensate for the difficulties investigators across the NIH have had with preserving, recovering and using animal models when many labs were working remotely. 3) And finally, in no other year has the core developed a single technology that will be so useful in manipulating brain genes and expression for neuroscience research in primates. 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. Mouse ESC (embryonic stem cell) lines that express the green fluorescent protein continue to be licensed. All new AAV vectors developed in and published) from the core facility will be available at Addgene.org