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20 Cards in this Set
- Front
- Back
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Conditional Knock Outs ?
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not remove of the whole gens but part of the chromosomal DNA to make sure that the gens is knocked up ( no active gene product)
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Conditional knock out in mice ?
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Introduce plasmid or transgene ( rescue molecule) and propagation of such molecule in the germ line and on so.
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How to keep the plasmid in the mice cell ?
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Have a resistant gene and grow it on it. but in the female embryo ? make it transgenic *DNA directly into the chromosome" and a second transgene to check on it
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How conditional knock out is different from single cell organism and complicated organism
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1. Complicated organism does nor have the genetic product at the stem cell level compare to unicellular organism.
2.Regulation of DNA is dependant on the location of the choromosome in which the gene land |
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Real conditional knock out in mice ?
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One allele will be removed at a particular condition. No rescuing is needed but heterozygous knock out ( remove it when you want)
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So, how do we do it ?
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1. Viruses and Cre-recombinase that can recombine DNA and shuffle pieces of DNA
2. DNA sites specific for recombinant loxP sites + recombinase => recombination (put it on both site of DNA so it would not do anything bad as it is a conditional survival |
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Really ? Based on which principle does these thing work ?
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Base on where and when the recombinase will be expressed as well as the placement of loxP. Tramsgene with recombinas on other mouse and how the promoter is out of the contral
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Types of promoters that can be used?
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Natural promoter ( specific region for a sequence)
Tissue specific promoter Stage specific promoter Inducible promoter/ stimulated by a drug ( don't have to keep mouse UDI ) |
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How to hunt to for these sucker ?
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Well, if a protein is expressed at a particular stage or tissue hen the promoter will be analysed ( element and expression such that enhancer, sites for co-activation)
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Procedures for this thang !
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Mouse 1: Insert LoxP site as on knock out
Mouse 2:Cre-mouse with the cre-combinase. ( one loxP site on one chromosome as knock out) Mate the two mouses. => Mendelian principles, one will have the right combination. |
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Why this technique is good?
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Ensure the transgenic construct behaves the way you want it. , rigourous
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What promoter and cre-combinase do again ?
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promoter: specificity
Cre-combinase: cutting at a certain spot |
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HOw to make a transgenic mouse ?
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1. Inject foreign DNA into the egg
2. Take the progeny and screen for the recombinant phenotype |
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Onco-gense ?
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Overly expressed genes that cause cancer. Dominant effect and can study and having a transgene
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Dominant negative ?
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the way nature use to inactivate the function of one gene and not removal of it. A natural or atifical mutant that affect the function of the normal genes.
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Give me one example that this will be helpful?
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Dealing with redundant fucntions due to uncertainity in phenotype is hard to use knock out (triple if you have 3)
=. inhibitory function trump them all. |
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What is irrigation ?
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Inhibit the function of GDPase by not producing GEF to create trafic jam. THis works as an typical inhibitory thing that explore spaces of dominnat negative
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Another example (thyrosine kinase receptor)
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Embedded on the cell membrane. work on cytoplasmic site and specific to hormone, Therer 2: receptor I and II . Both and kinase function and the phosporylation of both will cause massive sinaling pathway. But in case we have a truncated domain that is caused by mutation, the hormon will no longer binds. If there are more truncated domain => inefficient dimer. Can occur physiologicallly
ex. Dorsophlia 1 allele is mutant => not enough function due to the binary nature things => pathway is blockeded due to not enough signal produce |
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Gene knock down
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Degradation of DNA
Interference: remove the gene, remove the DNA or act on protein function. Creat short hairpins: produce mostly in cell culture and not in a womb. Creat small dsRNA that will be recognize by dicer |
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Procedure
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Get some obligonuclotide, clone it in plasmid and put it in cells. This construct has a linker region that will form a nice little loop. complimentary sequence that will adhere to each other
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