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
