Chapter 20 Experimental Systems Dr Capers In vivo

Chapter 20 Experimental Systems Dr. Capers

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 ○ 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

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)

Let’s talk about Covid-19 testing: https: //covidtestingproject. org/faq. html

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

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=uu. T 08 OT 3 w. Tc

Fusion producing hybridoma

Immunoprecipitation Agglutination

Enzyme-Linked Immunosorbant Assay - ELISA ○ How does the ELISA work? https: //www. youtube. com/watch? v=RRbuz 3 VQ 100

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 Ig. G I’m interested in looking at (HS-Ig. G I injected into mouse) - This can then be used in Western Blot (next slide)

How to do a Western Blot: https: //www. youtube. com/watch? v=Vg. Au. Z 6 d. BOfs

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

Watch this video: https: //www. youtube. c om/watch? v=EQXPJ 7 eees. Q

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

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

Thymidine uptake ○ Used to measure cell division in lymphocyte cultures

51 Cr 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

CRISPR-Cas 9 ○ Works in both in vitro and in vivo situations ○ This system evolved to provide bacteria protection against bacteriophages

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

Most common animal model for human research – mouse ○ Inbred strains reduce experimental variation

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

Transgenic animals

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

Knockout Mice – eliminating gene Watch this video: https: //www. youtube. com/watch? v=i. VY-Uki. Y fz. Y

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