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Principal Investigator: William Elkins
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $46,528,760
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Mouse Genetics and Gene Modification (MGGM) core has offered state-of-the-art CRISPR/Cas9 genome editing, embryo/sperm cryopreservation, rederivation of mouse lines, and ES and iPSCs services to thirty-six (36) NIAID investigators. The CRISPR/cas9 gene targeted animals were created on NOD ShiLtJ, C57BL6/J and C57BL6/N mouse background that saved almost one year for backcrossing to the investigators. Homozygous congenic mouse lines for CD45.1 and Thy1.1 markers were created as common resource for the NIAID investigators for competitive cell transplantation studies.
1)CRISPR cas9 genome editing: Completed sixteen (16) CRISPR/Cas9 gene KO and Gene KI projects including a) Bi-cistronic gene constructs using IRES and P2A peptide; b) generated complex multi-gene KO mice with two, three or four genes targeted simultaneously; c) improved CRISPR/Cas electroporation into embryos which has resulted in 70-80% gene editing efficiency in the newborn pups.
2)Generation of CD45.1 and Thy 1.1 congenic mouse lines: CD45.1 and Thy1.1 are two useful congenic markers for competitive cell transplantation study of hematopoietic stem and progenitor cells (HSPCs). The current animals available from NIAID-Taconic repository, have mixed genetic background which complicates the interpretation of results. Using CRISPR/cas9 gene editing, MGGM has created congenic mouse lines for CD45.1 and Thy1.1 on pure C57BL/6 Taconic and Jackson background as common NIAID resource. The mice have been crossed for multiple generations to generate homozygous lines. These mice are being used by the investigators from different institutes of NIH.
3)Cryopreservation of mouse lines: MGGM completed 170 projects for sperm/embryo cryopreservation and rederivation of mouse models, that included a) eighty-four (84) projects for sperm cryopreservation; b) fifty (50) projects for sperm QC; c) five (5) projects for embryo cryopreservation; d) three (3) for embryo QC and, e) twenty-eight (28) projects for rederivation of lines with the embryos from cryopreserved by MGGM or outside sources. MGGM significantly improved the conditions for in vitro fertilization (IVF) that resulted in high yield of embryos and reduced costs for females as embryo donors. We have also established vitrification method for cryopreservation of embryos and cryopreserved 250-350 embryo for each mouse line. Further, with the improved IVF, the efficiency of rederivation of lines also improved significantly. We have provided 10-30 pups for the rederived mice.
4)Vascularized cerebral organoids: MGGM has established systems for creating organoids using co-culture of progenitor cells (PCs) from E9.5 embryo with the pluripotent stem cells. Cerebral organoids generated by this approach contained all types of neuronal and glial cells including microglia and the endothelial cells. The system is being improved to develop organoids from human iPS cells.
5)NOD ShiLtJ ACE2 Humanized animals: ACE2 is the receptor to which SARS-Cov-2, the virus responsible for COVID19, binds to enter the cell. Animal models play a critical role in investigating the infection and disease progression as well as for the development of interventional approaches. Mouse in general is a poor model for SARS-CoV-2 infection because of difference of fifteen amino acids in ACE2 protein from human ACE2. Using successive CRISPR/cas9 gene knock-in editing, we have created mice in which the endogenous 15 AAs have been substituted with human AAs on a NOD ShiLtJ background as a potential model for patients with underlying medical conditions. These mice were then interbred to generate homozygous animals. The DNA sequence analysis has revealed correct insertion of the mutations.
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.
•15 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).
•1056 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.
•2167 formalin-fixed paraffin embedded (FFPE) tissue blocks made and slides (average) stained with hematoxylin and eosin (HE) for routine microscopic examination.
•13002 unstained slides.
•3,557 immunohistochemical slides including both established lab markers and dilution titration experiments for novel markers.
o3380 chromogenic, including 74 multilabel
o177 immunofluorescence, including 66 multilabel
•327 slides for in situ hybridization (including 87 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.
Terms: <0-4 weeks old><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><Aerobic><Affect><Allergy><Amino Acids><Animal Experimental Use><Animal Experimentation><Animal Model><Animal Models and Related Studies><Animal Research><Animals><Autopsy><B220><Backcrossings><Beds><Binding><Biological Terrorism><Biopsy Sample><Biopsy Specimen><Bioterror><Bioterrorism><Blood Precursor Cell><Board Certification><Body Tissues><Books><Breeding><CD45><COVID-19><COVID-19 infection><COVID-19 virus><COVID-19 virus infection><COVID19 infection><COVID19 virus><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><CV-19><Caring><Cas nuclease technology><Cell Body><Cell Transplantation><Cells><Cerebrum><Cessation of life><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Co-culture><CoV-2><CoV2><Cocultivation><Coculture><Coculture Techniques><Collaborations><Communicable Diseases><Complex><Confounding Factors (Epidemiology)><Confounding Variables><Congenic Mice><Contracting Opportunities><Contracts><Coronavirus Infectious Disease 2019><Cryofixation><Cryopreservation><DNA Sequence Analyses><DNA Sequence Analysis><Data><Death><Derivation><Derivation procedure><Diagnosis><Diagnostic><Diagnostic tests><Disease><Disease Progression><Disorder><Distress><Electroporation><Embryo><Embryonic><Emerging Communicable Diseases><Emerging Infectious Diseases><Endothelial Cells><Environment><Epidemiologic Confounding Factor><Equipment><Evaluation><Exclusion><Experimental Designs><Female><Fertilization in Vitro><Food><Formalin><GP180><Gene Modified><Gene Targeting><Generations><Genes><Genetic><Germ-Free><Glia><Glial Cells><Gnotobiotic><Gnotobiotics><H and E><HEPA Filter><HEPA Filtration><HEPA cleaner><Health><Hematopoietic Progenitor Cells><Hematopoietic stem cells><Hematoxylin and Eosin><Hematoxylin and Eosin Staining Method><Hortega cell><Human><Human Resources><Hypersensitivity><IACUC><IRES><Immunofluorescence><Immunofluorescence Immunologic><Immunohistochemistry><Immunohistochemistry Cell/Tissue><Immunohistochemistry Staining Method><In Situ Hybridization><Individual><Infection><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Insertion Mutation><Institutional Animal Care and Use Committee><Internal Ribosome Entry Segment><Internal Ribosome Entry Site><Investigators><Journals><KO mice><Knock-in><Knock-out Mice><Knockout Mice><Knowledge><Kolliker's reticulum><LY5><Label><Laboratories><Laboratory Animal Medicine><Laboratory Animals><Magazine><Manpower><Manuscripts><Medical><Medicine><Methods><Mice><Mice Mammals><Microglia><Microscopic><Microscopy><Mission><Modeling><Modern Man><Modification><Molecular><Molecular Interaction><Murine><Mus><NIAID><NIH><National Institute of Allergy and Infectious Disease><National Institutes of Health><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neuroglia><Neuroglial Cells><Neurons><Newborn Infant><Newborns><Non-neuronal cell><Nonneuronal cell><Null Mouse><Organism><Organoids><PTPRC><PTPRC gene><Pain><Painful><Paraffin Embedding><Pathogenesis><Pathologist><Pathology><Pathology Report><Patients><Peer Review><Peptides><Play><Pluripotent Stem Cells><Preparation><Prevention><Principal Investigator><Process><Progenitor Cells><Proteins><Protocol><Protocols documentation><Public Health><Publications><Publishing><Receptor Protein><Research><Research Activity><Research Personnel><Research Resources><Research Support><Researchers><Resources><Ribosome Entry Site><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 infection><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Scientific Publication><Services><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome related corona virus 2><Side><Sight><Slide><Source><Specialist><Specialty><Sperm><Spermatozoa><Staining method><Stains><System><T200><Technical Expertise><Techniques><Test-Tube Fertilization><Time><Tissue Banks><Tissue Collection><Tissue Embedding><Tissue repository><Tissues><Titrations><Training><United States National Institutes of Health><Veterinary Pathology><Virus><Vision><Work><Wuhan coronavirus><air filter><aminoacid><angiotensin converting enzyme 2><angiotensin converting enzyme II><animal experimentations><animal model development><assisted reproduction><blood cell progenitor><blood progenitor><blood stem cell><blood-forming stem cell><brain vascularization><cellular transplant><cerebral><cerebral vascularization><cold preservation><cold storage><comparative><congenic><coronavirus disease 2019><coronavirus disease 2019 infection><coronavirus disease 2019 virus><coronavirus disease-19><coronavirus disease-19 virus><coronavirus infectious disease-19><cost><depository><design><designing><develop therapy><electroporative delivery><embryo cryopreservation><embryo freezing><embryonic cryopreservation><embryonic freezing><experience><experiment><experimental research><experimental study><experiments><gene electrotransfer><gene modification><genetically modified><genome editing><genomic editing><gitter cell><hCoV19><hematopoietic progenitor><hematopoietic stem progenitor cell><hemopoietic progenitor><hemopoietic stem cell><hiPSC><high efficiency particulate air cleaner><high efficiency particulate air filter><high-efficiency particulate air purifier><human iPS><human iPSC><human induced pluripotent cell><human induced pluripotent stem cells><human inducible stem cells><iPS><iPSC><iPSCs><improved><in situ Hybridization Genetics><in situ Hybridization Staining Method><induced human pluripotent stem cells><induced pluripotent cell><induced pluripotent stem cell><inducible pluripotent stem cell><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><intervention development><knockin><living system><medical countermeasure><medical specialties><mesoglia><microglial cell><microgliocyte><microorganism><model of animal><mouse genetics><mouse model><murine model><nCoV2><necropsy><nerve cement><neuronal><new marker><newborn child><newborn children><novel><novel biomarker><novel marker><perivascular glial cell><personnel><pluripotent progenitor><posters><postmortem><preparations><programs><pup><receptor><repository><routine imaging><semen cryopreservation><social role><sperm cell><sperm cell cryopreservation><sperm cryopreservation><sperm freezing><spermatozoa cryopreservation><stem cells><success><technical skills><therapy development><treatment development><visual function><zoosperm>