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. Of significant note, the introduction of 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) has met with success and allowed for greater avenues of germ free studies. The Mouse Genetics and Gene Modification (MGGM) laboratory continued to provide state-of-the-art CRISPR/Cas9 genome editing, embryo/sperm cryopreservation and rederivation of mice services and expanded the program to generation of organoids from ES and iPSCs. Gene edited animals were created on the mouse background of investigators choice such as Swiss Webster, C57BL6J and C57BL6/N background that saved almost one year of backcrossing. Recently, MGGM has also created new congenic mouse strains for CD45.1 and Thy1.1 markers as common resource for NIAID investigators for the cell transplantation studies. CRISPR cas9 genome editing: Gene edited animals were created for; a) complex conditional gene KO (CKO) using large plasmids (>3.0kb); b) Bi-cistronic gene constructs using IRES and P2A peptide; c) gene KI with a multi-cistronic gene construct containing tdtomato, Cre and DTR separated by two P2A sequences knocked-in the Granzyme C gene; d) introduced new technique of electroporation of CRISPR/Cas reagents into intact embryos which has significantly improved the gene editing efficiency. Creation of new CD45.1 and Thy 1.1 congenic mouse lines: CD45.1 and Thy1.1 are two useful congenic markers for implantation to study immune cells in mouse. Over the years investigators have used animals with mixed genetic background from NIAID-Taconic repository. Using CRISPR/cas gene editing, MGGM has created new congenic mouse lines for CD45.1 and Thy1.1 on pure C57BL6 background from Taconic and Jackson labs. These lines will be available in the future to the NIAID investigators as a common resource. Establishment of organoid systems: MGGM has established systems for creating organoids using co-culture of progenitor cells (PCs) with the stem cells. Cerebral organoids generated by this approach contained all types of neuronal and glial cells including microglia and the endothelial cells. The system will be expanded to organoids for other tissues as well. Cryopreservation of sperm/embryo and rederivation of lines: MGGM completed over 150 projects for long term storage and preservation of mouse models, that included a) seventy-eight projects for sperm cryopreservation; b) twenty-three projects for sperm QC; c) two projects for embryo cryopreservation; d) forty-nine projects for rederivation of lines with embryos from cryopreserved 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. 25 new (not including ongoing active protocols from previous years) NIAID IACUC-approved animal research protocols requiring direct pathology support (i.e. routine microscopy, IHC/ISH, etc). 69 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). 621 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 and slides (average) stained with hematoxylin and eosin (HE) for routine microscopic examination. 12,500 unstained slides. 3270 immunohistochemical slides (3250 chromogenic, 25 immunofluorescence). 276 slides for in situ hybridization (including 78 labeled with an additional fluorescent marker). 9 scientific manuscripts with IDPS personnel as co-authors published in peer reviewed scientific journals, accepted for publication, and/or in the review process. *Note* A small percentage of NIAID investigators utilize an outside contract laboratory to process tissues and develop tissue blocks for routine microscopic (HE) evaluation. These tissue blocks and/or HE slides are transferred to the IDPS for evaluation and additional diagnostic tests (i.e. IHC/ISH, histochemical stains, etc), as needed. Although the microscopic assessment of slides and the subsequent pathology reports are generated by the IDPS pathologist, the