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Flies and Worms: Genetic Studies of Touch and Taste

Flies and Worms: Genetic Studies of Touch and Taste. October 23, 2003. Overview BLASTing genes Real Time PCR in situ hybridization Expression of proteins in cell culture Dominant-negative constructs RNAi Primary C. elegans cultures FRET imaging in C. elegans Notes on electrophysiology.

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Flies and Worms: Genetic Studies of Touch and Taste

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  1. Flies and Worms: Genetic Studies of Touch and Taste October 23, 2003

  2. Overview BLASTing genes Real Time PCR in situ hybridization Expression of proteins in cell culture Dominant-negative constructs RNAi Primary C. elegans cultures FRET imaging in C. elegans Notes on electrophysiology

  3. BLAST The Website: www.ncbi.nlm.nih.gov/BLAST/ 24 October 2003 We will be removing the obsolete 'nph-blast_report' web service. Read more... DisclaimerPrivacy statementAccessibilityThis page is valid XHTML 1.0.

  4. BLAST What you can learn from sequence homology I have this gene but I don’t know its function-- are there other genes that look like my gene? What do they do? I think I have a gene that serves a particular function-- do other genes that serve similar function look like my gene? There is a gene family that I want to study-- are there other genes in this family that have not been studied? How similar are different genes in this family from my gene? Are there certain regions that are very similar across this family? What function does my gene serve in other species?

  5. Real Time PCR Quantifies changes in gene expression by means of fluorescent markers Best for showing relative changes in gene expression Uses reference sample and experimental samples Relies on fluorescent reporter that binds specifically to dsDNA, emitting fluorescence only when bound RT-PCR is performed on both samples As PCR product increases, fluorescence increases (linear relationship) Samples can be compared to determine relative quantities Ambion Tech Notes www.ambion.com.techlib/tn/85/857.html and 81/813.html

  6. In Situ Hybridization Dioxygenin Used as alternative to radioactive labeling Probe is anti-sense RNA labeled with Dioxygenin Labeled RNA binds to mRNA in sample Apply antibody to dioxygenin Apply stain for antibody Determine location/absence of mRNA for your gene in sample www.fruitfly.org/about/methods/cytogenetics.html

  7. Dioxygenin “Digoxigenin can be incorporated at either terminus, at both termini, and/or within the sequence. Eurogentec produces these oligonucleotides by using either a dT-amino modification or a C6-amino modifier to create an attachment site for the digoxigenin moeity” http://usa.eurogentec.com/code/en/page_02.asp?Page=77

  8. Cell Culture Studies Basic Protocol: Select cell line Clone gene of interest into vector (includes tags, promotors) Transfect gene into cells Induce gene expression (image to see fluorescent gene expression or immuonstain for specific proteins) Lyse cells and spin to remove cell debris Run over purification column to isolate protein Remove from column Run on gel-- use antibody for protein or tag to confirm

  9. Creation of Dominant-Negatives Basic Principle: Create mutant that expresses the wild-type gene, but this gene cannot perform its function due to specific interference by another construct Examples: Binding by inactive protein domain Overexpression of inactive subunit of complex Blocking of active sites on protein (direct or indirect) Caveats: Theoretical construct does not always work as d-n Must be sure that effect seen is due to interaction with your gene

  10. RNAi Usually potent and specific Double-stranded more effective than sense or anti-sense single stranded (Injected single strands probably rapidly degraded) dsRNAi mimics loss of function phenotype Injection produces decrease in endogenous mRNA May act at level of chromatin structure or transcription Crosses cellular boundries (injection in head of one animal can result in interference in progeny) Fire et al. (1998) Nature 391: 806-811

  11. RNAi Limitations: If members of a gene family share similar sequence, construct may interfere with multiple genes If gene is expressed at low levels, not all genes or all cells will be influenced Fire et al. (1998) Nature 391: 806-811

  12. C. elegans Primary Cultures Enables access to individual cell types for functional and molecular studies Isolated cells differentiate into cells which would be found in L1 larva (as yet, no postembryonic development) Uses strains with cell-type specific fluorescent markers to identify desired cells Fluorescence Activated Cell Sorting (FACS) Plated cells express cell-specific markers Effective techniques for functional characterization: Patch clamp Whole cell recordings dsRNA knock down of specific gene expression Christensen et al. (2002) Neuron 33:503-514

  13. C. elegans Primary Cultures Christensen et al. (2002) Neuron 33:503-514 Figure 2. Micrographs of Cultured Embryonic Cells Expressing Cell-Specific GFP Reporters(A) myo-3::GFP expression in cultured body muscle cells. myo-3 encodes a myosin heavy chain isoform expressed in body muscles. The myo-3 reporter strain expresses two GFPs with peptide signals that target them to either the nucleus (arrows) or mitochondria (arrowheads). Inset: immunolocalization of UNC-54 myosin in body wall muscle cells. Cultures derived from myo-3::GFP-expressing worms were fixed and incubated with anti-UNC-54 and a Cy3-conjugated (red) secondary antibody. Yellow indicates overlap of GFP and Cy3 fluorescence. (B) Cultured mechanosensory neuron expressing mec-4::GFP. mec-4 encodes a degenerin-type ion channel subunit expressed largely in neurons that respond to gentle body touch. (C) Cultured cholinergic motor neurons expressing unc-4::GFP. unc-4 encodes a homeodomain transcription factor. Inset: immunolocalization of synaptotagmin in motor neurons. Cultures derived from unc-4::GFP-expressing worms were fixed and incubated with anti-synaptotagmin and a Cy3-conjugated (red) secondary antibody. unc-4::GFP, anti-SNT-1, and DAPI fluorescence are shown in green, red, and blue, respectively. Yellow indicates overlap of GFP and Cy3 fluorescence. (D) Cultured neuron expressing opt-3::GFP. OPT-3 is a H+/oligopeptide transporter expressed in glutamatergic neurons.

  14. C. elegans Primary Cultures Christensen et al. (2002) Neuron 33:503-514 Figure 2. Micrographs of Cultured Embryonic Cells Expressing Cell-Specific GFP Reporters(E) Combined DIC and fluorescence micrograph of an unc-119::GFP-expressing neuron. unc-119 encodes a novel protein that is expressed in all neurons and eight head muscle cells. (F) Combined DIC and fluorescence micrograph of an unc-4::GFP-expressing cholinergic motor neuron physically interacting with a body wall muscle cell. (G) Lateral view of a transgenic larval worm showing expression of unc-4::CFP (green) in A-type motor neurons and acr-5::YFP (red) in B-type motor neurons in the ventral nerve cord. Anterior is to left. (H) unc-4::CFP and acr-5::YFP are expressed in separate sets of neurons in vitro after 5 days in culture. All scale bars are 10 μm.

  15. Chameleon What is FRET? Fluorescence Resonance Energy Transfer Emission wavelength of one molecule is excitation wavelength for another molecule When the 2 molecules are in very close proximity and the first molecule is excited, emission spectra from the second molecule can be detected If the molecules are not in close proximity, emission spectra from the first molecule will be detected

  16. Chameleon How does chameleon work as a calcium sensor? Miyawaki et al. (1997) Nature 388:882-887

  17. Chameleon Advantages/Disadvantages Advantages: Precise targeting Easy to image Does not interfere with normal function of cell Clean and specific Able to detect a wide range of Ca++ concentration Can be quantified with high spatiotemporal resolution Disadvantages: Nonzero baseline-- rare associations would be hard to detect Miyawaki et al. (1997) Nature 388:882-887

  18. Notes on Electrophysiology Neurons maintain their resting potential near reversal potential for K+ (around -80 mV) Na+/K+ pump results in high extracellular Na+ and low extracelluar K+ But, neurons are leaky Adding large amounts of K+ externally upsets the ionic balance, changes the reversal potential for K+ and depolarizes the cell The measurement in cell recordings is the flow of Cl- ions on and off a silver-plated reference electrode

  19. Additional Resources National Center for Biotechnology Information http://ncbi.nlm.nih.gov FlyBase http://flybase.bio.indiana.edu WormBase http://www.wormbase.org C. elegans genetics http://elegans.swmed.edu/genome.shtml Worm Anatomy and Behavoir http://www.wormatlas.org

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