Comparative Medicine Infectious Diseases- Bethesda

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

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. Of significant note, this year the CMB introduced a novel combination of "germ free" IVC racks in conjunction with a change station equipped with a SteriMist decon system to allow for multiple small cage studies to be performed at the same time. The Mouse Genetics and Gene Modification (MGGM) laboratory has provided state of the art CRISPR/ Cas9 genome editing, embryo/sperm cryopreservation and rederivation of mice services to thirty-three (33) NIAID investigators. Several genome-edited, gene knock-(KI), gene knockout (KO) and conditional cre-lox knockout (CKO) animals, were created using ss-oligonucleotides (ss-ODNTs) and large DNA plasmids and two stage IVF mediated gene KI strategy. CRISPR cas9 genome editing: Using CRISPR/Cas editing, a) genome edited animals were completed for three highly complex conditional gene KO (CKO) projects using large plasmids (>3.0kb) containing lox sequences; b) A highly specialized split-cre approach was used to create two sets of animals with two cell specific promoters each controlling one part of the cre recombinase for cell specific deletion. c) Several gene KO animals were created for complete gene KO using 2 sgRNAs; d) In another complex gene KI approach, simultaneously two ODNTs and two sgRNAs were injected to create CKO animals with both Lox sequences using a small molecule SCR7 as an alternate approach to two step flox procedure. Embryonic stem (ES) cells: Using morula aggregation technique, several chimeric animals were created using Lin28 gene KO cells received from Mirimus LLC company. Five animals were created by aggregation of these cells with FVB/N morula stage embryos which exhibited 100% contribution of the Lin18 cells. In another project BV2 cells were gene edited for CARD9 gene via CRISPR mediated gene targeting. Cryopreservation of sperm and embryo and rederivation of lines: In a major achievement, MGGM has created a second location for the cryopreserved samples at Bldg. 50. Overall, 152 projects were completed for sperm & embryo cryopreservation and rederivation of mouse lines to buildings 50, TB3, 14BS. These projects included a) eighty-seven projects for sperm cryopreservation; b) twenty-six projects for sperm QC; c) two projects for embryo cryopreservation; d) two projects for embryo QC by creating live pups; e) Thirty-seven projects for rederivation of lines with embryos cryopreserved either by MGGM or outside sources. The Infectious Disease Pathogenesis Section (IDPS), utilizing a collaborative and integrative One-Health approach, directly supports NIAID investigators, programs, and collaborators through the incorporation of pathology-based, animal model development and use, and IACUC-approved research to facilitate and improve diagnoses, treatments, preventions, and medical countermeasures of infectious diseases in humans. The IDPS conducts comprehensive postmortem examination (i.e. necropsy) and tissue collection training on laboratory animal species; and provides complete microscopic tissue evaluation utilizing a wide array of diagnostic, molecular, and special studies to support spontaneous and experimental disease pathogenesis research primarily involving significant and/or emerging public health threats. Equipped with and working alongside other core services, our board-certified veterinary pathologist(s) and highly specialized technical team is committed to providing concise and dependable results to collaborating researchers as well as publication-worthy summary findings (to include photomicrographs) in order to continue to advance the missions and vision of the NIAID. The IDPS provides subject matter expertise and technical guidance in veterinary pathology related activities in support of researchers and collaborators across the NIAID (and on occasion other Institutes within the NIH); as well as to the Comparative Medicine Branch through provision of diagnostic pathology services ensuring a thriving animal colony for use in biomedical research. 24 NIAID IACUC-approved animal research protocols requiring direct pathology support (i.e. routine microscopy, IHC/ISH, etc) 26 interim and/or final pathology reports provided to NIAID research and investigators complete with summaries, scoring tables, and and/or images of routine microscopic and other special studies findings (e.g, immunohistochemistry, histochemical stains, ISH, etc) 269 individual animal IDs submitted from research protocols or submitted for (e.g., unexpected death); submissions include single tissue/biopsy specimen to a partial and/or complete sets of tissue from a partial and/or complete postmortem examination (i.e. necropsy), respectively 1,997 formalin-fixed paraffin embedded tissue blocks made 12,500 slides (average) stained with hematoxylin and eosin (HE) for routine microscopic examination 2,160 immunohistochemical slides 226 slides for in situ hybridization