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Causes of cell injury
- Oxygen deprivation
- Physical
- Chemical
- Infectious
- Immune Reactions
- Genetic
- Nutritional
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Players in cell injury
- ATP depletion
- Calcium
- membrane permeability
- mitochondrial damage
- Oxygen & reactive oxygen species
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A useful plasma contains these 3 features
- origin of replication for DNA polymerase
- antibiotic resistance gene
- convenient site for DNA insertion
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What is subcloning
recombining DNA fragments into a new plasmid vector
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What is transformation
introducing a plasmid into E. coli such that it becomes a permanent fixture
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Why is an antibiotic resistance gene incorporated into the recombinant plasmid?
To identify which bacteria took up the plasmid
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Steps of PCR
- Denature DNA with heat
- add primers to template DNA
- add DNA polymerase to synthesize in a 5' to 3' direction
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Which type of vaccine replicates in the host
modified live
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Steps in creating a DNA vaccine
- 1. subclone gene for immunogenic protein (insert downstream from eukaryotic promoter)
- 2. Transform e. coli with vector
- 3. inject plasmid into the animal
- 4. plasmid is taken up by muscle cells
- 5. host cell expresses protein and antibodies are formed
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Advantages of a recombinant vaccine
- safety (can't return to virulence)
- DIVA (differentiate infected from vaccinated animals)
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West Nile virus gene put into a yellow fever virus is an example of
chimeric flavivirus
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What enzymes serve as scissors and paste for recombinant molecules?
- scissors = restriction enzymes
- paste = DNA ligase
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how do you perform PCR on RNA?
Convert it to a DNA template first with reverse transcriptase
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What is the Recombitek WNV vaccine?
canarypox vectored vaccine
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What is Prevenile?
yellow fever virus chimera vaccine
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What is Innovator WNV?
formalin inactivated vaccine
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Why wouldn't e. coli be a good host for producing a rabies glycoprotein vaccine?
E. coli cannot glycosylate proteins
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What is the difference between a chimera and a vector vaccine?
- Vector: DNA is added to the genome
- Chimera: DNA replaces a portion of the genome
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What is a subunit vaccine?
recombinant protein
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Explain the concept of a recombinant protein
a gene encoding the valuable protein is inserted into some type of host (expression system) and that host makes large amounts of the protein
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What is needed for a PCR? (5)
- Template DNA
- polymerase (heat stable)
- nucleotide triphosphates
- primers
- buffers
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How is bovine somatotropin made by recombination?
BST gene is inserted into a plasmid with a prokaryotic promoter and e. coli expresses it
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How are transgenic mammals produced?
inject the transgene into a one celled embryo, gene is taken up and expressed
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Define euploidy
chromosome count is normal
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Define aneuploidy
abnormal chromosome count
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define monosomy
a missing chromosome
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Define trisomy
3 copies of a chromosome instead of 2
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Define triploidy
triple the haploid number
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How do you calculate the diploid chromosome number of a hybrid species?
Add the haploid numbers of each species
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What are the 3 rules of x inactivation?
- 1. all but one X is functionally inactive
- 2. inactivation is random with respect to parental origin
- 3. inactivation is a permanent change (except during gamete formation)
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What is reciprocal translocation?
breakage and refusion with exchange of parts of a chromosome
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What are two types of translocations?
reciprocal translocation and centric fusion
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What is the chromosome complement of a mare with Turner's syndrome? What is the resulting phenotype?
63 X, underdeveloped reproductive tract
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What is the diploid chromosome number of a chimera (goat & sheep)?
cells are either 60 (sheep) or 54 (goat)
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Both parents are heterozygous for a autosomal dominant disease. How many of the offspring are affected?
75%
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Both parents are heterozygous for an autosomal recessive disease. How many of the offspring are affected?
25% (50% carriers)
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How are x linked mutant genes expressed in heterozygous females?
They are carriers
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Can females heterozygous for hemophilia develop disease even though they are carriers?
If most of the cells in the liver came from mutation on the active x chromosome, yes.
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What is penetrance?
How well does the genotype match the phenotype
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What is an example of incomplete penetrance/dominance?
Dexter cattle are autosomal dominant (Aa), but the aa genotype results in a lethal phenotype (bulldog calf).
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Give an example where a different mutations in the same gene result in very different phenotypes
androgen receptor mutations result in complete inactivation, testicular hypoplasia, or no effect at all
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What is an example of heterozygote advantage?
Sickle cell anemia - immune to malaria
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What are two examples of founder effect?
BLAD - neutrophil adhesion deficiency - traced back to 1 bull - autosomal recessive
HYPP is a voltage gated Na+ channel mutation traced back to Impressive
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What are multifactorial diseases?
Diseases that are familial but do not cleanly fit mendelian inheritance - affected by genetics and environment
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What are the proportions of poodles that are normal when the parents are PDA x PDA (100%), PDA x 1st gen relative (75%), and PDA x Normal (50%)?
- 100% = 17% normal, 66% with disease
- 75% = 33% normal, 45% with disease
- 50% = 78% normal, 10% with disease
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What is liability? What is threshold?
- liability is the "dose" of bad gene + bad environment
- threshold - must be reached to show signs of disease
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What is a morphological diagnosis?
communication tool used to convey the cause and significance of tissue damage
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What does 3DATP stand for and which ones are most important?
- degree, duration, distribution, adjective, tissue, process
- T&P
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What is an etiologic diagnosis?
cause and tissue process
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What is the outcome of ATP depletion
shift to glycolysis, intracellular P rises, pH drops
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What happens has Ca2+ builds up in the cell
widespread enzyme activation
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What happens during mitochondrial damage?
mitochondrial permeablility transition, cytochrome C leakage, programmed apoptosis
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Oxygen and ROS damage is from
normal cellular metabolism and inflammatory cells
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Oxidative damage results in
membrane, protein, & nucleic acid damage
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Reversible cell damage is characterized by:
loss of volume control & cell swelling
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What causes the cell to swell during hypoxic conditions?
- ATP depletion results in loss of Na/K/ATPase pump
- Na flows in toward gradient, proteins can't get out = increased osmolarity
- water flows in
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What are myelin figures?
injured membrane (whorls)
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