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22 Cards in this Set
- Front
- Back
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Title: Dream Caused by the Flight of a Bee Around a Pomegranate a Second Before Awakening
Artist: Salvador Dali |
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Use of Studying Animals
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-study etiology & pathogens of a dz that affects humans
-discovery therapies/cures -obtain scientifically valid research, the conditions associated w/an experiment must by closely controlled: manipulating only one variable while keeping other constant & then observing consequences of that change |
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Reasons for Choosing Animals
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-similarity to humans (genetics, anatomy, physiology)
-unlimited supply -ease of manipulation -adequate number of subjects (statistically significant tests) |
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Rodents Commonly Used B/c...
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-rats, mice, gerbils, guinea pigs, hamsters
-majority of genetic studies have employed mice -genomes similar to humans -availability -ease of handling -high repro rates -relatively low cost of use -others used = fruit flies, zebra fish, bakers yeast |
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Mutations Causing Dz
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1 Null - mutation leads to loss of gene production
2. hypomorphic - mutation leads to less gene product 3. dominant negative - mutation leads to expression of aberrant protein |
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Methods to Induce Human Dz in other Organisms
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-most animal models do not contract the same dz
-2 methods: -non-directed and mutation driven -directed and dz driven |
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Non-Directed & Mutation Driven
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-uses radiation & chemicals to cause mutations
-common technique = large scale mutation screen -randomly make mutations in animal model's genomes -animals screened for phenotypes that are similar to human dz -useful for identifying new genes & pathways that contribute to dz |
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Large-Scale Mutation Screen
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-x-rays often cause large deletion & translocation mutations that involve multiple genes
-N-ethyl-N-nitrosourea (ENU) |
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N-ethyl-N-nitrosourea (ENU)
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-ENU treatment linked to mutations w/in single
genes, such as point mutations -ENU can produce mutations w/many different types of effects, such as loss & gain of function -frequently employed in screens in model organisms such as zebra fish |
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Directed & Dz Driven
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-Palmiter & Brinster (1982) - transgenesis: allowed targeted genetic manipulation in an animal
-Common techiques include: -transgenesis -single-gene knock-outs & knock-ins -conditional gene modifications -chromosomal rearrangements |
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Transgenesis
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-adding foreign genetic information to nucleus of embryonic cells
-first mouse transfers (gene) performed in 1980 - not optimal -developed a way to increase the size of the DNA fragments used by cloning them in yeast or bacterial artificial chromosomes (YAC's of BAC's) -contain regulatory sequences necessary for normal gene expression -more comparable to the endogenous gene |
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First Gene Mouse Transfers
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-foreign DNA was incorporated into only a small percentage of embryos and was inconsistently passed to the next generation
-small transgenes were inserted into random sites in the genomes & depending on their location they were not always expressed |
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Two Methods of Transgenesis
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-injecting the foreign DNA directly into the embryo
-use a retroviral vector to insert the transgene into an organism's DNA |
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Knock-Out V.s. Knock-In Mice Differences
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Knock-Out:
-carry gene that has been inactivated -loss of function Knock-In -produced by inserting transgene into exact location -gene is over expressed |
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About Knock-Out/Knock-In Mice
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-both target mutation to a specific gene locus
-useful if a single gene is shown to be the primary cause of a dz -goal: specific mutation is inserted into the endogenous gene - conveyed to next generation through breeding -embryonic stem cells (ES) required |
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Embryonic Stem Cells (ES)
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-can contribute to all cell lineages with injected into blastocysts
-can be genetically modified & selected for the desired gene changes -homologous recombination creates the mutations -a process that physically rearranges two homologous strands of DNA for the exchange of genetic material |
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Making a Knock-Out Mouse
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-selectable marker (Neomycin resist) added to target sequence
-target vector added to gene seq. from black mouse -target vector knocks out copy of mouse target gene in ES -DNA in some cells take up the target vector -only cells that have taken up the vector survive when neomycin is present -cells w/knock out mutation injected into developing white mouse embryo -resulting chimera mouse - contains mix of own cells & heterozygous knock out cells -bred w/non-transgenic white mouse -of all offspring, black mouse has KO gene - can pass on to offspring |
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Conditional Gene Modification
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-many KO & KI mice have similar, if not identical, phenotypes to human patients - good models for human dz
-many disorders occur late in life -mutations can profoundly affect development & cause early death -generate mutations in specific tissues & at different stages of development, including adulthood -2 different types of genetic alterations needed |
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Two Types of Genetic Alterations
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1. Conditional Vector - an "on switch for mutations
-Cre recombinase enzyme -under control of tissue-spec. promoter X 2. Specific sites (called loxP) - inserted on either side of a gene (Y), or part of a gene, that encodes a certain component of a protein that will be deleted -cross the mice -when Cre expressed in tissue X - gene Y deleted -specific mutation at spec. time in spec. tissue |
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Chromosomal Rearrangements
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Cre/loxP conditional mutations - creation of models for complex human dz's involving chromosomal rearrangements in selected tissues
-using site-specific mutations -include chromosomal deletions, duplications, inversions, & translocations |
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Mouse Models Effective as Models for Human Genetic Dz's
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-over 1000 mutant strains exist & most mutants are models for inherited genetic dz's
-mutating same gene in mice that is responsible for the human condition for about 100 genes -most cases: replicate many of the corresponding human dz phenotypes -FEW, if any, replicate all symptoms -neurodegenerative dz's - cognitive defects -mice not capable of expressing some cognitive human dz symptoms |
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Need for Better Animal Models?
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-non-human primates - physiology closer to humans
-downside = technical difficulties, ethical considerations, & additional costs to perform transgenesis in primates -transgenic model of Huntington's dz was recently developed using Rhesus macaque that replicated some of the characteristic pathologies |
