The Biomedical Research Infrastructure (BRI) is an underground two floor, 120,000 square foot, 27,000 cageanimal facility. This state-of-the-art automated facility is in the courtyard of the Biological Laboratories, whichplaces it adjacent to the Northwest Building. The BRI houses a Genome Modification Facility (GMF) in an 800square feet procedural room in the BRI with additional animal holding space for 1,000 cages in adjacentrooms. This room is equipped with 4 vibration free microinjection stations, micro-needle fabrication equipment,4 banks of tissue culture incubators for embryo/ES cell culture, 3 tissue culture hoods, 3 laminar flow hoods forpathogen free minor surgeries, 12 stereomicroscopes of which 2 will be equipped with GFP optics andteaching video, automatic liquid nitrogen storage facilities, and computers.The GMF as a whole is governed by faculty committee of four individuals: Andrew McMahon (Molecular andCellular Biology, Committee Chair), Joshua Sanes (CBS), and Kevin Eggan (HSCI). The committee overseeseffective running of the GMF.The GMF staff consists of the following. The director, Manfred Baetscher, PhD, has broad experimental experience of genetic modification ofmice and many years of managerial experience. He participates directly in production of geneticallymodified strains, remains current on emerging genetic approaches, and advises investigators onappropriate genetic strategies for their experiments. Transgenic research assistants: Two assistants, both with proven experience in either or bothpronuclear injection of DMA and blastocyst injection and small animal surgery. Tissue culture technicians: A full time technician, soon to be hired will perform all aspects of ES cellculture, ES cell electroporation and clonal selection and DMA preparation forgenotyping. Animal husbandry: Two animal care technicians conduct all support functions for embryo generationand biopsy preparation. Administrative assistant: This individual is responsible for all ordering, billing and coordination betweeninvestigators and GMF.Finally, it is worth noting that the BRI will also contain behavioral testing suites equipped for phenotypicanalysis of genetically modified mice, including models of neurological and psychiatric diseases. These will besupported from other sources, so are not described here, but it is our intention to integrate them with the GMF.The GMF provides the following services: Production of transgenic mice by pronuclear injection of DMA constructs in plasmids, BACs, or YACs ES cell targeting and injection to generate 'knock-out' and 'knock-in' mice Cryopreservation and storage of embryos and sperm for valuable strains not in current use Mouse strain resuscitation by in vitro fertilization and intracytoplasmic sperm injection (ICSI) Re-derivation of mouse strains by embryo transfer to render them pathogen-free The development of lentiviral vector based transgenes Consultation on genetic model creation Plans to offer gene targeting in embryonic stem cells and validation of DNA vectors and feeder linesWe also hope the GMF will provide new functionality that we will add if this grant is funded, circumventing abottleneck that many users of genetically modified mice encounter. There are a dozen or so strains that arehighly useful for a broad variety of neuroscience applications. Most are publicly available but the waiting time toobtain them collaborators or stock centers (e.g., at Jackson Laboratories) is often 6 months or more, and theexpense is considerable. Yet, few laboratories can afford to maintain stocks permanently of lines that mayneed only once every year or two. Lines in this category include: (a) Mice in which specific neuronalpopulations are marked with GFP or one of its spectral variants (e.g., motoneurons in YFP-16, layer 5pyramids in YFP-H, very small numbers of forebrain neurons in GFP-M, or inhibitory interneurons in GAD65-GFP). (b) Mice in which expression of a reporter is conditional on expression of ere recombinase (e.g., Z/EG,Z/AP and thy1-stop-YFP). (c) Mice in which ere or flp recombinase is expressed in the neural tube generally(e.g., Nestin-Cre) or in specific populations of neurons (e.g., CaM kinase-Cre) or only following activation bytamoxifen (e.g., CAGS-CreER). So, we propose to maintain small colonies (approximately 4 cages of 4 maleseach) of each of about a dozen lines. The lines will be chosen by the Steering Committee. In most cases, themice are already available in at least one user laboratory. Then, when any Harvard neuroscientist requiresone of the lines, we will be able to provide two males within a week, and then replenish our own supply bybreeding. We believe this repository will greatly decrease the barrier to use of valuable strains, thereby makingpossible risky or pilot experiments that would otherwise be prohibitively expensive or unduly slow.
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