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 Mouse Genetics and Gene Modification (MGGM) Section provided state of the art CRISPR/ Cas9 genome editing, embryo/sperm cryopreservation and rederivation of mice services to over thirty (30) NIAID investigators. Several different tools of genome editing such as ss-oligonucleotides (ss-ODNTs), large DNA plasmids, long single stranded (lssDNA), nickase mediated gene KI and two stage gene KI mediated by IVF, were applied to create gene knock in (KI)/knockout (KO) and conditional cre-lox knockout (CKO) animals. CRISPR cas9 genome editing: MGGM has made significant improvement in CRISPR/Cas editing and successfully completed a) four highly complex conditional gene KO (CKO) projects using large plasmids (>3.0kb) and lssDNA with cre-lox sequences; b) another gene KI project completed with large plasmid containing Cre-recombinase gene; c) four projects for complete gene KO using 1 and 2 sgRNAs; d) Another complex gene KI project where two mutations were knocked-in the genome simultaneously using ODNTs. Embryonic stem (ES) cells: Several gene targeted NBC17 ES cell clones for Lin28 gene were received from Mirimus. Culture conditions were established for the propagation of these cells to create chimeric animals for germline transmission of Lin28 mutations. Cryopreservation of sperm and embryo and rederivation of lines: For long term storage and preservation of mouse models, a total of 149 projects were completed that included a) Ninety-nine projects for sperm cryopreservation; b) twelve projects confirmation for sperm QC; c) five projects for embryo cryo; d) three projects for embryo QC by creating live pups; e) Thirty projects for rederivation of lines with embryos either cryopreserved by MGGM or outside sources. The Infectious Disease Pathogenesis Section (IDPS) supports NIAID investigators in the execution of many activities related to investigative animal research studies including, but not limited to: animal study design; necropsy instruction and assistance; creating and refining tissue sample collection protocols; ensuring that investigators are well-informed of animal model-related anatomy, physiology, and appropriate pathology terminology. 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 many functional changes and improvements to the services provided to the NIAID investigator community. The IDPS is a full-service histopathology laboratory with full tissues-in-slides-out capabilities. In addition, the IDPS uses state-of-the-art digital pathology slide scanning technology which allows easy sharing of data both internally and abroad and facilitates performing semi-quantitative image analysis on a diverse set of samples. 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. To date, the IDPS continues to generate both traditional and innovative pathology data sets for investigators; many of these data sets are associated with publications that are currently in progress, have been submitted for publication or, have been accepted in highly respected, peer-reviewed, journals including The New England Journal of Medicine, Nature, Nature Immunology, Plos Pathogens, and Journal of Infectious Diseases, to name a few. Our main objectives are to continually increase the laboratorys efficiency, expertise, and scientific contribution without compromising quality; retain the IDPS quest for innovation by continued implementation of cutting-edge technology; and uphold a commitment to maintaining the highest standard of pathology research support and investigation for the NIAID investigator community. The IDPS most significant contributions have, in large, been related to our ability to help highlight the pertinent scientific findings and details, which exist in in the gap between the generation of a hypothesis and those assays executed at the benchtop. Ultimately, we help investigators examine the broader and more basic questions of pathogenesis related to lesser understood disease processes and help clarify the significance of newly identified disease-specific features using animal model systems and archived human tissue samples