Causes of cell injury
- Oxygen deprivation
- Immune Reactions
Players in cell injury
- ATP depletion
- membrane permeability
- mitochondrial damage
- Oxygen & reactive oxygen species
A useful plasma contains these 3 features
- origin of replication for DNA polymerase
- antibiotic resistance gene
- convenient site for DNA insertion
What is subcloning
recombining DNA fragments into a new plasmid vector
What is transformation
introducing a plasmid into E. coli such that it becomes a permanent fixture
Why is an antibiotic resistance gene incorporated into the recombinant plasmid?
To identify which bacteria took up the plasmid
Steps of PCR
- Denature DNA with heat
- add primers to template DNA
- add DNA polymerase to synthesize in a 5' to 3' direction
Which type of vaccine replicates in the host
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
Advantages of a recombinant vaccine
- safety (can't return to virulence)
- DIVA (differentiate infected from vaccinated animals)
West Nile virus gene put into a yellow fever virus is an example of
What enzymes serve as scissors and paste for recombinant molecules?
- scissors = restriction enzymes
- paste = DNA ligase
how do you perform PCR on RNA?
Convert it to a DNA template first with reverse transcriptase
What is the Recombitek WNV vaccine?
canarypox vectored vaccine
What is Prevenile?
yellow fever virus chimera vaccine
What is Innovator WNV?
formalin inactivated vaccine
Why wouldn't e. coli be a good host for producing a rabies glycoprotein vaccine?
E. coli cannot glycosylate proteins
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
What is a subunit vaccine?
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
What is needed for a PCR? (5)
- Template DNA
- polymerase (heat stable)
- nucleotide triphosphates
How is bovine somatotropin made by recombination?
BST gene is inserted into a plasmid with a prokaryotic promoter and e. coli expresses it
How are transgenic mammals produced?
inject the transgene into a one celled embryo, gene is taken up and expressed
chromosome count is normal
abnormal chromosome count
a missing chromosome
3 copies of a chromosome instead of 2
triple the haploid number
How do you calculate the diploid chromosome number of a hybrid species?
Add the haploid numbers of each species
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)
What is reciprocal translocation?
breakage and refusion with exchange of parts of a chromosome
What are two types of translocations?
reciprocal translocation and centric fusion
What is the chromosome complement of a mare with Turner's syndrome? What is the resulting phenotype?
63 X, underdeveloped reproductive tract
What is the diploid chromosome number of a chimera (goat & sheep)?
cells are either 60 (sheep) or 54 (goat)
Both parents are heterozygous for a autosomal dominant disease. How many of the offspring are affected?
Both parents are heterozygous for an autosomal recessive disease. How many of the offspring are affected?
25% (50% carriers)
How are x linked mutant genes expressed in heterozygous females?
They are carriers
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.
What is penetrance?
How well does the genotype match the phenotype
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).
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
What is an example of heterozygote advantage?
Sickle cell anemia - immune to malaria
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
What are multifactorial diseases?
Diseases that are familial but do not cleanly fit mendelian inheritance - affected by genetics and environment
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
What is liability? What is threshold?
- liability is the "dose" of bad gene + bad environment
- threshold - must be reached to show signs of disease
What is a morphological diagnosis?
communication tool used to convey the cause and significance of tissue damage
What does 3DATP stand for and which ones are most important?
- degree, duration, distribution, adjective, tissue, process
What is an etiologic diagnosis?
cause and tissue process
What is the outcome of ATP depletion
shift to glycolysis, intracellular P rises, pH drops
What happens has Ca2+ builds up in the cell
widespread enzyme activation
What happens during mitochondrial damage?
mitochondrial permeablility transition, cytochrome C leakage, programmed apoptosis
Oxygen and ROS damage is from
normal cellular metabolism and inflammatory cells
Oxidative damage results in
membrane, protein, & nucleic acid damage
Reversible cell damage is characterized by:
loss of volume control & cell swelling
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
What are myelin figures?
injured membrane (whorls)