L3 - READING FOR TUESDAY Ch 11 p651-660 661-688 First...

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READING FOR TUESDAY Ch. 11 p651-660, 661-688 First problem set is available on the web
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Fluorescence Microscopy Fluorescent molecules absorb light at one wavelength and emit it at another-longer wavelength. Uses filters to allow only light of a given wavelength in and out. Can detect specific proteins or other molecules in cells and tissues. Fluorescein (emits green), rhodamine (deep red) are molecules that can be chemically coupled to proteins to detect their localization indirectly GFP, isolated from jellyfish is a protein (encoded by a gene) that has intrinsic fluorescents Fluorescent molecules can be bleached
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Indirect immunofluorescence allows one to follow the localization of proteins in a cell
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Indirect immunofluorescence
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Section of an E11.5 mouse spinal cord illustrating a gradient of netrin protein (red immunofluorescence) in the path of commissural axons extending to the floor plate at the ventral midline (neurofilament M, green immunofluorescence). Immunofluorescence shows that netrin-1 protein, as distinct from netrin-1 mRNA (see in situ hybridization) is expressed in the floor plate
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1) You read the old literature and realize that Ramon y Cajal was a really smart guy and on to something. 2) You devise and assay based on Cajal’s ideas that you can use to biochemically purify an axon guidance activity 3) You biochemically purify the activity with the help of two post-docs in your new lab at UCSF 4) You molecularly clone the gene and make antibodies against the protein 5) You prove the gene you cloned is in the right place at the right time to mediate the activity 6) You prove it has the activity using your in vitro culture 7) You knock the gene out in mice to prove that loss of the gene’s activity causes axon guidance defects 8) At this point, other people in other organisms are working on the gene. You collaborate with scientists working in C. elegans to clone the receptors in your lab
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clone the vertebrate homologs of Dcc and Unc5h Function of Netrin-1 is Evolutionarily Conserved UNC-6 Netrin-1 Nematode: C.elegans Netrin-1 receptors were cloned in C. elegans by identifying mutants with similar uncoordinated phenotypes: Unc6 (netrin), Unc40 (DCC) and Unc5 (Unc5H) DCC UNC5H
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RNAi can be used to knock-down the function of genes Fire and Mello 2006 Nobel Treat cells in tissue culture or animals with siRNA (small interfering RNAs) or a plasmid that expresses them.
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RNAi can be used to knock-down the function of genes Fire and Mello 2006 Nobel Double-stranded RNA is cut into short pieces (siRNA) by the endonuclease Dicer (natural pathway) OR an investigator introduces siRNA into the cell (synthetic pathway).
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