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Immunology

Chapter 20 Experimental Systems Dr. Capers. Immunology. In vivo Involve whole animal In vitro Defined populations of immune cells are studied under controlled lab conditions. Study of immune system requires suitable animal models

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Immunology

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  1. Chapter 20 Experimental Systems Dr. Capers Immunology

  2. In vivo • Involve whole animal • In vitro • Defined populations of immune cells are studied under controlled lab conditions

  3. Study of immune system requires suitable animal models • For vaccine development – is the animal model susceptible to the disease? • Mouse most often used • Inbred strains reduce variation caused by differences in genetic backgrounds • 20 or more generations of brother-sister mating • Have to abide by IACUC guidelines

  4. Cell Culture Systems • Cells are cultured and studied • Needs specialized media • Growing cells that are used to being in a multicellular organism, need specific growth factors, etc. • Can be used for: • Testing effects of contaminants on immune cells • Testing drugs (can be done before trying in vivo) • Producing monoclonal antibodies • Cell line • Cells that have been transformed – propagate indefinitely (cancerous cells)

  5. Antibody Generation

  6. Polyclonal antibodies • Immunizing animal (mouse, rabbit) or human with antigen one or more times • Taking blood samples, purifying the antibodies from the serum • Results in a mixture of antibodies directed towards variety of different epitopes • Disadvantages: • Ill-defined cross-reactivities with related antigens • Range of cross-reactivity to desired antigen might vary from bleed to bleed • Animal might die, causing you to start over

  7. Monoclonal antibodies • Product of single, stimulated B cell • Supply of antibody specific for one epitope • Uses: • Can be specific for specific target cells and conjugated to toxins • More sensitive/specific ELISAs • How to produce monoclonal antibodies: https://www.youtube.com/watch?v=uuT08OT3wTc

  8. Fusion producing hybridoma

  9. Immunoprecipitation and Agglutination-based Techniques

  10. ImmunoprecipitationAgglutination

  11. Antibody assays based on molecules bound to solid-phase supports

  12. Enzyme-Linked Immunosorbant Assay - ELISA • How does the ELISA work? • https://www.youtube.com/watch?v=RRbuz3VQ100

  13. Protein Biochemistry • Gel Electrophoresis • SDS-PAGE • SDS is a detergent, binds to proteins and destroys tertiary and secondary structure • Proteins can be separated according to molecular weight • Separation of antibody classes (different heavy chains, separation of light and heavy chains) • Run IgG I’m interested in looking at (HS-IgG I injected into mouse) • This can then be used in Western Blot (next slide)

  14. How to do a Western Blot: https://www.youtube.com/watch?v=VgAuZ6dBOfs

  15. Immunofluorescence imaging techniques

  16. Fluorescent Technology • Lots of different kinds • Green fluorescent protein • Isolated from bioluminescent jellyfish • Can be used to visualize live cells

  17. Flow Cytometry and cell sorting

  18. Watch this video: https://www.youtube.com/watch?v=EQXPJ7eeesQ

  19. Flow cytometry and cell sorters have important clinical applications • Detect/classify leukemias • Prognosis of AIDS • Identifying stem cell populations in bone marrow transplants • Need increasingly sophisticated software

  20. Magnetic bead cell sorting • Conjugate antibodies to magnetic beads, put in column

  21. Cell cycle analysis

  22. Thymidine uptake • Used to measure cell division in lymphocyte cultures

  23. Assays of cell death

  24. 51Cr Release Assay • Used for measuring Tc and NK cell activity • Target cells are incubated first with Chromium-labeled sodium chromate which is taken up by cells • If the target cells are killed, the chromium is released and can be measured

  25. CRISPR-CAS9

  26. CRISPR-Cas9 • Works in both in vitro and in vivo situations • This system evolved to provide bacteria protection against bacteriophages

  27. WHOLE ANIMAL EXPERIMENTAL SYSTEMS

  28. Animal research is subject to federal guidelines that protect nonhuman research species • Animal Welfare Act 1966 • All institutions performing research on vertebrates must have IACUC • Institutional Animal Care and Use Committee • Guide for the Use and Care of Laboratory Animals

  29. Most common animal model for human research – mouse • Inbred strains reduce experimental variation

  30. Adoptive Transfer • Lymphocyte cells isolated from donor • Put into recipient who has been irradiated • Allows one to see the function of an isolated cell population

  31. Transgenic animals

  32. Limits with transgenic mice: • Transgene is randomly integrated within genome • Might insert in area of DNA that is not transcriptively active • Or might disrupt vital genes causing other problems • So researchers will sometimes use knock-out mice • Replace gene with truncated, mutated or altered form of gene

  33. Knockout Mice – eliminating gene • Watch this video: https://www.youtube.com/watch?v=iVY-UkiYfzY

  34. Gene Transfer • Knockout mice • Replace normal gene with mutant allele • Steps: • Isolation and culture of embryonic stem cells from mouse blastocyst • Generation of desired altered form of gene • Introduction of that altered gene into the cultured embryonic stem cells • Injection of those stem cells into mouse blastocyst • Implantation into psuedopregnant female • Mating of offspring herterozygous for the altered gene to eventually get homozygous knockout mouse

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