Comparative Medicine Infectious Diseases- Bethesda

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

CMB Research Support Specialists provide technical expertise and support to NIAID Principal Investigators, assisting them with true cage to benchside support. Both pathology and technical proficiency resulted in numerous co-authorship opportunities. The CMB has successfully maintained a gnotobiotic breeding and study facility, which consists of bio-exclusion units designed to keep the mice from becoming colonized with any adventitious microorganisms. Germ-free mice are free of all aerobic and anaerobic organisms with the possible exception of endogenous viruses. Breeding colonies and mice on study are maintained in isolators provided with HEPA-filtered air and autoclaved food, bedding, and supplies. Strict SOPs are followed to maintain the mice in a germ-free state. The mission of the Mouse Genetics and Gene Modification (MGGM) Section at CMB/NIAID is to provide highly sophisticated CRISPR Cas9 mediated genome editing in embryos and embryonic stem (ES) cells as well as related services such as: a) cryopreservation of sperm b) cryopreservation of embryo for long term storage of valuable mouse lines and c) rederivation of lines from cryopreserved embryos and sperm. During this year, MGGM made significant progress in CRISPR/cas9 mediated genome editing including gene knock in (KI) via oligonucleotides the and large DNA plasmids. Two microinjection specialists hired last year are now well trained and our turnover rate for the projects is about 8-10 weeks with 50-100% efficiency of indels for CRISPR cas9 mediated genome editing. Two microinjection units, one each at Twinbrook III and Bldg. 50, are fully operational. The building 50 component provides MNV and helicobacter free animals to the NIAID investigators. A tissue culture lab is fully set at Twinbrook III for ES cell systems for gene knock in/knockout via homologous recombination and by CRISPR/cas9 system. Over 30 different NIAID investigators have used MGGM services. CRISPR cas9 mediated genome editing: Gene editing by CRISPR/cas9 was completed for 10 independent projects. Three of the projects involved gene KI using oligonucleotides and double stranded plasmid. Two projects were carried out using 100-160nt oligonucleotides whereas another KI project involved gene KI via approx. 8kb plasmid. Transgenic mouse production: Several lines of transgenic mouse were created by direct microinjection of DNA into embryos Cryopreservation of lines via cryopreservation of sperm and embryo: o MGGM has successfully completed 33 sperm cryopreservation projects which were then tested by sperm QC to generate pups and confirmed by genotype analysis o MGGM successfully completed 10 embryos cryopreservation projects. Approx. 250-350 embryos were cryopreserved for each project and the embryos were tested by QC to generate pups and confirmed by genotype analysis Rederivation of mouse lines: Over 25 different projects were completed for rederivation of lines with the embryos either cryopreserved by MGGM or imported from outside sources. Embryonic Stem (ES) cells: Three new independent ES cell lines were established from C57BL6 ES embryos using retinol mediated activation of endogenous machinery. One of the male cell line, was used to generate chimeras. The microinjection of these cells into blastocysts and morula aggregation has generated chimeras with more than 80% ES cell contribution. The chimeric mice are currently being tested for germline transmission. The Infectious Disease Pathogenesis Section (IDPS) supports NIAID investigators in the execution of many activities related to animal studies including, but not limited to: animal study design; necropsy instruction and assistance; tissue sample collection protocols; and education on the anatomy, physiology, and appropriate pathologic terminology and description for study specific diseases/agents. The IDPS also aids in the interpretation of data and the incorporation of these data into manuscripts for publication. The IDPS continues to improve the quality of pathology support given to NIAID investigators and the level of communication/collaboration between the IDPS and the laboratories within NIAID. The IDPS has made a number of functional changes and improvements to the services provided to the NIAID investigator community. The IDPS has recently transitioned to a full-service histopathology laboratory with full tissues-in-slides-out capabilities. In addition, the IDPS has acquired a state-of-the-art digital slide scanning system that allows easily sharing of data abroad and the ability to use a number of semi-quantitative analytical tools to generate data for investigators. These technologies will now give the IDPS the ability to process a wide range of tissue types and perform various analyses within our laboratory space. This year, the IDPS has generated several traditional and innovative pathology data sets for investigators for which publications are currently in progress, have been submitted for publication or, have been accepted in highly respected peer-reviewed journals including Nature, Nature Immunology, Plos Pathogens, and Journal of Infectious Diseases, just to name a few. Our main objectives are to continually increase the laboratorys growth and efficiency while not compromising quality; keep the IDPS advanced by continued implementation of cutting-edge technology; and to continue to fine-tune the performance of the IDPS histology laboratory in efforts to maintain the highest standard of pathology research support for the NIAID investigator community.