LATG Immunolgy and mouse breeding part 3 Flashcards
Dna-> ES CELLS ->blastocyst, so that DNA is incorporated into many cells.
Embryonic Stem Cell-mediated Transgenics
Embryonic stem (ES) cells are undifferentiated so can become:
Any cell in the body
ES cells are harvested from__ then grown in ____.
Inner mass of blastocyst, culture
Many 129 strains of mice, including __ have embryos too delicate for pronuclear injection. Therefore ____ is used for those.
Balb/c’s, ES trangenesis
Es cells can can be introduced using ___, where a solution of ES cells and DNA are given an electric shock. The shock opens pores in the cellular and nuclear membranes so DNA can move through. Es cells selected for DNA presence through resistance of antibiotics. Cells w/ transgene injected into blastocyst. (ES cell mediated transgenesis method)
Electroporation
Transgenic lines usually remain WT, outbred to be _____ bc transgenes can be inserted ___ and interrupt another gene.
Heterozygous, anywhere
____ strains are commonly chosen for genetic engineering bc easier to see ____ changes when compared to transgenic mouse.
Inbred, phenotypic
Pups are ____ to confirm incorporation of transgene. Only ___ of pups will incorporate gene.
Genotyped, 20%
Each mouse w/ incorporated gene is new _____ of a line. Insertion of gene is ___ for each founder
Founder, different
Founders should ___ be bred w/ each other. They are bred with____.
Not, WT
If genes pass from founder to offspring, the transgene has been integrated into_____.
Germ cells
If transgene is in germ layers the offspring:
Will have genes in the same place on the genome as their parent.
Hemizygous:
Transgenic animals only have 1 copy of gene. Instead of the normal paired gene.
2 hemizygous animals bred produce:
25% homozygous for transgene. 50% hemizygous for transgene. 25% no copy of transgene.
If you want to remove/change/ replace a gene you need:
Gene targeting
In gene targeting, ES cells (from blastocyst) are grown in the presence of _____. Surviving cells have taken up DNA w/ resistance.
Antibiotics
In gene targeting, ES cells are used bc:
millions can be screened for the rare homologous event. Cells that test + for homozygosity are injected into the blastocyst.
Gene-targeting results in _____mice b/c some blastocyst cells are from dame mouse and some are from ES cells.
Chimera
Injecting cells from a mouse w/ a different _____ ____ allows researchers to follow which cells were from which group of cells.
Coat color, also makes which offspring are chimeric obvious.
ES cells tend to be (gender). If so they have (gender) embryo, and offspring.
Male, male.
If ES cells, which are (gender) result in a (gender) embryo, offspring may be one of 3 things:
Male, female (XX). : hermaphrodite, XXY sterile female, XY male
Germ-line transmission:
Targeted gene mutation in the germ cell layer of parent, to be transmitted to offspring.
Percent color of chimeric mice can be used to estimate:
Take-up of ES cells by offspring.
Pups that receive Es cell chromosomes will have a copy of:
Mutated gene in all cells of the body.
(Gender) Chimeras are preferred over (gender) for breeding.
Male/female (boooooo)
CRISPER/Cas9 uses ___ to target and cut at specific place in the genome.
Enzymes
CRISPER is naturally occurring ___ immune system for bacteria.
Anti-viral
In CRIPSER, when recognizing a foreign nucleic acid, DNA is targeted for cutting by associated nuclease_____.
Cas 9
With CRISPER, guided RNA is used to bring___ to correct spot of DNA. Either DNA is degraded at cut creating a ___ ____ or a new DNA fragment is incorporated, creating a ____ _____.
nuclease, knock-out, knock-in
W/ CRISPER, if any 1 allele is mutated, animal is considered:
Mosaic
In CRISPER a__ ___ is usually done at 2 weeks of age.
tail biopsy
Backcrossing:
Breeding mutant mice with WT mice of the desired strain to dilute the genome of the ES cells. (Beyond the desired trait).
Backcrossed generations are given what type of numbering system?
N1, N2, N3, etc.
Once there are ten backcrossed generations, the mouse is considered ___ ____to the inbred strain, except for the desired modification. This is considered a___ __.
genetically identical, cogenic strain
Recovering a mouse line:
When thawed and put into a pseudopregnant female.
Sperm that is cryopreserved results in ___ offspring. Not optimal for loves that have 2+ modified genes or need to be ___.
Heterozygous, homozygous
Means “well-known life”. Germ-free/axenic are one type.
Gnotobiotic
Defined Flora (DF):
Germ free, but with gut bacteria for digestion.
Specific pathogen free are ___ gnotobiotic. Have normal flora….
but free of certain pathogens.
This isolator marked the beginning of modern gnotobiotic research. By Dr. Phillip Trexler (Notre Dame).
Trexler flexible-film
Demand increased for axenic/gnotobiotic animals after NIH launched ____ _____ _____ in 2007. Characterized human microbiome.
The Human Microbiome Project
Flexible isolators are made of clear ___ or ___ plastic film.
Polvinyl or polyurethane
Semirigid isolators may be made of ___ ___ material on all but one side, which has a flexible material.
Impact-resistant
Isolators usually have built-in ___ ___ to allow manipulation w/o contamination.
Neoprene gloves
Isolators also have parts with ___ & ____ ___ so items can pass through.
Inner and outer seal
Isolators go through how many wks of testing before animals introduced?
3-6 consecutive weeks
Breeding isolators hold _ cages.
12+
Experimental isolators hold _ cages.
3-5
Mice are ___ each time the port to isolator is opened.
Tested
Supply cylinder:
Stainless steel entry port. Has holes w/ filters to allow steam/gas to pass through and sterilize.
Transfer sleeve:
Flexible plastic tube btwn entry port and sterile container or isolator. Acts as barrier lining as things are passed through into supply cylinder. Is wiped down with sterilant before use.
Laminar flow cabinet:
Provides cabinets or racks with filtered/sterile air. Removes airborne microorganisms. Animals more accessible here than in isolator.
Axenic:
Completely free of all microorganisms
Ways to sterilize:
Steam autoclave, peracetic acid, chlorine dioxide, hydrogen peroxide gas plasma, gamma irradiation, ethyline oxide, dry heat, membrane filtration
Gnotobiotic mice achieved by:
Hysterectomy redrivation or embryo transfer+ barrier rearing to get microorganism-free mice.
Problems with axenic mice:
Thinner intestinal walls, poor muscle development, tissue enzymes are lower, smaller lymph nodes, peyers patches and thumuses. Cecal torsion more common due to large colon size.
Fun facts axenic animals:
Longer life span, derived from hysterectomy and foster to axenic mom or embryo transfer to axenic surrogate.
After hysterectomy derivation a surrogate from a line with a different color coat than pups is important…
so pups don’t get confused.
Axenic mice must be check for ___ and ___ bc these can pass the placenta barrier.
microplasma, pasteurella
Axenic rat pups must be checked for ___ ___ ____ and ____ ____ ___.
Lymphocytic choriomeningitis virus, hilhaun rat virus.
Axenic animals are given what type of gut bacteria?
A cocktail of Symbiotic/ non-pathogenic
Characteristics of self are attributed to macromolecules on the cell surface called the
Major his to compatibility complex (MHC) proteins
Histo means:
Tissue
His to compatibility means:
Tolerance of immune system for body tissues.
The amount of response an antigen can produce.
Antigenicity
Main parts of the immune system where leukocytes are formed:
Thymus, bone marrow, liver, spleen, lymphatic system.
Innate immunity,
1st line of defense
These are all part of the innate immunity:
Saliva, tears, nasal secretions. Also contain lysozyme, which lyses bacterial walls.
The 2nd line of defense in the INNATE IMMUNITY are _______ in the blood stream.
Complement proteins
Complement proteins are mostly produced in the ___, but also by ___ and ____.
Liver, macrophages and monocytes
Compliment proteins are activated by MHC of ______ cells and/or _____ surface molecules.
Foreign, pathogen
Complement proteins can puncture _____ of foreign cells causing lysis.
Membrane
Interferons:
Chemicals that carry out communication among cells. 3 types: alpha, beta, gamma
Interferons are produced by many cells, including:
Immune cells, endothelial cells, and fibroblasts
These molecules job is to 1)interfere with viral replication, 2) heighten immune response, 3) stimulate activity of immune cells.
interferons
Macrophages and granulocytes (chiefly neutrophils) are part of the innate immunity. They:
Respond to patterns of molecules on other cell membranes that are not ‘self’. Exude lysosomal vesicles containing digestive enzymes to attack invaders. Also initiate phagocytosis.
Phagocytosis:
Vesicles made inside cell containing foreign material/antigen. Vessicle connects to lysosome and is lysed.
Phagocytes:
Present parts of the lysed antigen on their cell surface. Cells that do this are the Antigen Presenting Cells (APC).
APC’s attach to MHC’s on the____ cell, where other cells can recognize antigen and mount their response.
phagocytosing
This system is both cell-mediated and antibody mediated responses.
Acquired immune system
Cell - mediated immune response:
specialized lymphocytes (T cells) stimulate immune response. T-cells submit cytokines to stimulate immune response in other cells.
These cells release cytotoxic molecules that destroy foreign cells, cancer cells and injured cells.
T Cells
These cells stimilate B-cells, including B-cells turning into plasma cells.
T cells
When B-cells turn into plasma cells, they then produce:
Antibodies
Antibody mediated immunity is also known as:
Humoral immune response(body fluids)
Plasma cells secrete antibodies that neutralize or destroy infectious agents. This is part of what immune response?
Antibody mediated
3 steps of acquired immunity:
1) recognition, 2) amplification, 3) resolution
1st step of acquired immune system, recognition:
Occurs when B or T cells encounter APC. MHC + antigen+T cell receptor. T cell is ‘turned on’ and moves into amplification stage.
2nd step of acquired immune system, amplification:
Pathogen specific T cells proliferate. B cells mature into plasma cells. (Which increase antibody titer). T cells communicate with other molecules and cells through cytokines and chemokines. Chemokines regulate cell traffic through lymphatic system. Induce T cell proliferation, produce inflammation response.
Resolution, step 3 of acquired immunity:
Threat is eliminated and immune system scales back in response to protect healthy tissues. Anti-inflammatory response cytokines and growth factors are produced, causing antigen-specific cells to die. B-cell specific to antigen that did not turn into plasma cell are stored in lymphatic system. Are now memory B-cells. Likewise memory T-cells are made.
Granulocytes:
neutrophils, eosinophils, mast cells and basophils
Phagocytose or release lysosome contents
neutrophils
These cells release histamine.
Mast cells and eosinophils.
APC’s that trap antigen on surface and “present” them in lymph nodes.
Dendritic cells
B cells come from where in birds?
Bursa of Fabricius
Helper cells secrete:
cytokines
____ are ____ which are glycoprotiens that bind to antigens. This is also known as ___-___ complex.
antibodies, immunoglobulins (Ig), antibody-antigen complex.
Base of antibody is Fc fragment. Binds to receptors of:
leukocytes
Passive immunity:
antibodies from 1 animal to another. Occurs naturally when antibodies cross placenta or colostrum. Is only temporary.
Antiserum injection is another example of what? Lasts a few weeks.
passive immunity
Primary immune response:
the immune response occurring on the first exposure to an antigen, with specific antibodies appearing in the blood after a multiple day latent period.
The amount of antibodies present is:
a titer
These types of vaccinations are preferred for immunization because they stimulate the best immune response.
Live, attenuated viruses
If a virus has a high mortality, this type of vaccine is preferred.
dead, more stable and cannot become virulent.
killed mycobacterium tuberculosis, water-in-oil emulsion, severe reaction, painful, can only be used on 1st immunization.
Freuds Adjuvant complete
No bacterium, water-in-oil emulsion, moderate reaction, non-painful, can be used for boosters as well as 1st immunization.
Freuds Adjuvant incomplete
4 types of immune diseases:
Immunodeficiency, immune hypersensitivity, autoimmunity, chronic immune complex diseases.
Immune system fails to provide adequate response to some antigens. Classified as primary or secondary deficiency diseases.
Immunodeficient
Primary immunodeficiency:
innate error of metabolism or inherited genetic disease. ex: lacking thymus, certain Ig’s cannot be synthesized, cell mediated response does not work correctly.
Secondary immunodeficiency:
is acquired over time or due to disease or outside factors. ex: AIDs (acquired immunodeficiency syndrome) caused by HIV. Rids body of helper T cells ad microphages. SIV, FIV
Lowered immune responsiveness increases supsceptibility to infection/disease. Can be caused by genetics, radiation, or drug. ex: cyclosporin A inhibits T cell activation.
Immunosuppression
Overreaction of immune system to certain antigens. eX: allergies, asthema, autoimmune.
Immune hypersensitivity
Immune system does not recognize own cells as self.
Autoimmune disease
Chronic immune complex disease:
Antibodies stay in the blood longer than normal after infection and begin to attach to other tissues, such as the kidneys, lungs and joints. This causes swelling and damage.
Graft vs. Host Disease (GVHD)
When host recognizes the transplant as foreign.
Subclinical:
When disease shows no obvious symptoms.
No nucleus or organelles. Cell wall is proteoglycans or simple cell membrane.
Bacteria
Bacteria that have an large external cell-wall that stains purple-blue.
gram positive.
Bacteria that have a thin wall and stain pink.
gram negative.
Morphological characteristics are:
toxin? does it spore? shape? appendages? cillia? flagella? capsule?
Physiological characteristics:
oxygen? temperature? pH?
cocci=
round
cocobacilli=
ovoid
bacilli=
rod-like
spiral=
rods w/ twist ex:vibrio, spirillum, spirochete
pleomorphic=
appears in more than one shape
Rickettsia=
rarely seen in lab, varies as cocci or rods.
Made up of polysaccharides and proteins. Jello consistency. Can be in gram + or gram - bacteria.
capsule
Are 2 relevant types of bacteria that form spores.
Bacillus and clostridium
Spores:
genetic material in a tough outer coating
C.Piliformes
toxin-> enteritis->Tyzzers disease ->lethal for gerbils, foals.
most bacteria grow best at what temperature?
37 degrees celcius.
Most bacteria grow best at what pH range?
6.5-7.5
Streptococcus pheumonia is tested for in what media?
sheeps blood agar
Differential agar:
many different types of organisms can grow on it but reactions of media are discernably different. ex: by color.
DNA virus families.
pox virus, parvo virus, herpes virus, rota virus.
Largest most complex group of viruses. Many are zoonotic. Skin disease, severe diseases. ex: small pox, mouse pox, monkey pox, orf
pox virus
small virus. likely resistant to breakdown in environment. Wide range of symptoms in different species. ex: GI symptoms in dogs, brain malformation in neonatal cats, ammonia in NHPs and scrotal hemmorage in rats. MVM (minute virus of mice) highly contagious infection of T and B lymphocytes.
parvo virus
Causes sores/ulcers. B virus is zoonotic. DNA . Macaques can shed to humans even if asymptomatic. can cause encephalitis in humans. Can be fatal to both humans and NHPs.
herpes
These pathogens can be used as vectors to induce the creation of desired genome sequence. Vaccinia and adenovirus are both used for this.
virus
Orthomyxoviruses, paramyxoviruses, coronaviruses, arboviruses.
RNA viruses
RNA viruses that can cause lung disease.
Orthomyxoviruses and paramyxoviruses
Influenza is this type of virus and can infect many species.
Orthomyxoviruses
A paramyxoviruse that can infect NHPs w/ mild symptoms.
measles
These viruses also cause many respiratory illnesses including SARS (severe acute respiratory syndrome) in humans.
Coronavirus
Is the most significant mouse virus (corona virus) b/c even non symptomatic mice can have a severe immune supression, decreased lymphocyte production , decreased phagocytic activity.
Mouse hepatitis virus (MHV)
A highly contagious corona virus for rats. Affects eyes and lymphatic system.
Sialodacryodenitis virus (SDAV)
Arthopod borne viruses part of the togavirus family. Mosquitoes, ticks, lice carry to different animals.
Arboviruses
Eastern and Western equine encephalitis, yellow fever (NHPs) and West Nile are all examples of what type of virus?
Arboviruses
Prions
protein and amyloid plaque. Mad cow disease (bovine spongiform encephalopathy or BSE)
Scrapie, in sheep and goats is caused by:
prions.
These are sometimes called “slow viruses” b/c the incubation is more than 18mos, longer than the lifespan of many lab animals.
Prions
These are used as the prion encephalitis model.
Hamsters
This is currently the only way to get rid of prions.
Incineration
one-cell fungi are called:
yeasts
Molds are microscopic multicellular organisms w/ branching tubules called:
hypha
A group of hypha are called:
mycelium
Mycoses are:
diseases caused by fungi.
Microsporum, tricophyta, epidemophyten can cause this. Usually appears on head or chest of animal.
Ringworm
