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) core has provided valuable services in state-of-the-art CRISPR/Cas9 genome editing, embryo/sperm cryopreservation, rederivation of mouse lines, and ES and iPSCs to thirty-two (32) NIAID investigators. The CRISPR/Cas9 gene targeted animals were created on C57BL6/J and C57BL6/N mouse background that saved almost one year for backcrossing to the investigators. 1) CRISPR/Cas9 genome editing: Completed twelve (12) gene KO and gene KI projects including a) Bi-cistronic gene constructs using IRES and P2A peptide; b) generated complex multi-gene KO mice using four RGS genes, RGS1, RGS 2, RGS 13 and RGS18, targeted sequentially to create double and triple gene KO animals. The conditions were modified to improve CRISPR/Cas9 electroporation into embryos for high efficiency gene KI with one or two oligonucleotides to target more than one site simultaneously. 2) Propagation of homozygous CD45.1 and Thy 1.1 congenic mouse lines: CD45.1 and Thy1.1 are useful congenic markers for competitive bone marrow cell transplantation of hematopoietic stem and progenitor cells (HSPCs). Currently used C57BL6/SJL congenic mice have mixed genetic background which complicates the reproducibility and interpretation of the results. The MGGM, has created congenic mouse lines via CRISPR/Cas9 gene modification for CD45.1 and Thy1.1 on pure Taconic and Jackson C57BL/6 background. The mice from both genetic backgrounds and mouse strains have been crossed to create homozygous lines. These mice are being provided to the investigators from different institutes of NIH. Further, approx. 500 embryos from all the strains have been cryopreserved for protection and distribution of lines. 3) Cryopreservation of mouse lines: MGGM completed 169 projects for sperm/embryo cryopreservation and rederivation of mouse models, that included a) eighty-four (84) projects for sperm cryopreservation; b) twenty-one (21) projects for sperm QC; c) thirteen (13) projects for embryo cryopreservation; d) twelve (12) for embryo QC and, e) thirty-nine (39) projects for rederivation of lines with the embryos from in-house cryopreserved and outside sources. Conditions were improved for in vitro fertilization (IVF) that resulted in high yield of embryos and reduced number females as embryo donors. The conditions for cryopreservation of embryos via vitrification were improved further. Approximately 250 embryos for homozygous lines and approx. 350 embryos from heterozygous lines were cryopreserved for long term storage. With the improved IVF conditions, we provide 10-30 pups for the rederived mouse lines with reduced number of egg donors. 4) Thicket Rat ES cells: Earlier we had created novel induced pluripotent stem (iPS) and embryonic stem (ES) cell lines from thicket rat for the preservation of the colonies. The cell lines are being propagated for characterization and for investigating the pluripotency of the cell lines. The ES cells will be transduced with GFP vectors for visualization. The pluripotent cells will be injected into tetraploid mouse embryos to check their potential for the derivation of pure ES cell derived embryos. 5) NOD ShiLtJ ACE2 Humanized animals: Using successive CRISPR/cas9 gene knock-in editing, earlier we created humanized NOD ShiLtJ mice in which 15 AAs of mouse ACE2 gene were substituted with human AAs, the receptor for SARS-COV-2, as potential model for SARS-COV-2 infections in patients with underlying conditions. The homozygous animals were tested for SARS-COV-2 infections. Our initial studies in collaboration with Dr. Gunjan Arora from SVC facility have shown that the humanized NOD ACE2 mice are resistant to the infection with Omicron strain. These mice are being tested for infection with other variants of SARS-COV-2. 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, immunologic, and allergic diseases in humans. The IDPS conducts comprehensive postmortem examination (i.e. necropsy) and tissue collection training on laboratory animal species; provides complete microscopic evaluation of tissue 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; and is positioned to support other new approach methods, NAMs (e.g. organoids), as the science progresses. 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. • 39 active NIAID IACUC-approved animal research protocols requiring direct pathology support (i.e. routine microscopy, IHC/ISH, etc). • 68 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). • 771 individual animal IDs submitted from research protocols or submitted for pathology (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. • 1544 formalin-fixed paraffin embedded (FFPE) tissue blocks made (approx.), and slides (average) stained with hematoxylin and eosin (H&E) for routine microscopic examination. • 9000 unstained slides (approx.). • 2907 immunohistochemical slides including both established lab markers and dilution titration experiments for novel markers. o 2596 chromogenic o 284 with only heat-induced epitope retrieval (HIER) applied o 27 immunofluorescence, including 11 multilabel • 289 slides for in situ hybridization (including 44 labeled with an additional fluorescent marker). • 18 scientific manuscripts with IDPS personnel as co-authors published in peer reviewed scientific journals, accepted for publication, and/or in the review process (also includes three poster presentations and one book highlighting work performed by IDPS personnel). • *Note* A small percentage of NIAID investigators utilize an outside contract laboratory to process tissues and develop tissue blocks for routine microscopic (H&E) evaluation. These tissue blocks and/or H&E 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 numbers reported above do not account for this additional workload produced by the contract laboratory.