fluorescent protein reporters and fluorescence technology josh leung james weis february 18th, 2010...
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![Page 1: Fluorescent Protein Reporters and Fluorescence Technology Josh Leung James Weis February 18th, 2010 Bio 1220, Gary Wessel](https://reader030.vdocuments.us/reader030/viewer/2022020111/56649cea5503460f949b5a81/html5/thumbnails/1.jpg)
Fluorescent Protein Reporters and Fluorescence Technology
Josh LeungJames WeisFebruary 18th, 2010Bio 1220, Gary Wessel
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Fluorescence: How does it work?
Fluorescence vs Phosphorescence◦ -Time delay –
microsecond vs minPhoton absorbed
→ photon released◦ -One photon vs two
photonsGFP, other
technologies mainly use fluorescence
Jablonski Diagram
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What is Fluorescence used for?In Biology
◦Fluorescent proteins and fluorophore tagging cellular integrity, endocytosis, exocytosis,
membrane fluidity, protein trafficking, signal transduction, enzymatic activity, genetic mapping, etc.
◦Fluorescence Microscopy◦DNA Microarrays (test for gene expression)◦DNA Sequencing◦Fluorescence Recovery After Photobleaching◦Fluorescence-Activated Cell Sorting
Other uses include fluorescent lighting, flame tests, etc.
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Advantages of Fluorescence TechnologyTagging a target molecule
◦In vivo detection◦Reliable (even down to one
molecule)◦High fidelity and specificity◦Identify multiple target molecules
simultaneouslyDevelopment of new imaging
techniques◦Also detect more types of targets
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Fluorescence MicroscopyShine light →
fluorescence → detection
Separate weaker fluorescence from the excitation light using filters
Limit of detection determined by the darkness of the background (lack of noise, etc)
↓Specimen
Camera↑
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Fluorescence Microscope
C. Elegans Nervous System Cell Division
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Fluorescence Microscope
Mammalian Cells (DNA is blue, microfilaments are green)
Endothelium Cells(Triple fluorescence staining of endothelium cells from a pulmonary artery)
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Opossum Kidney Cortex Epithelial Cells (OK Line)
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Human Cervical Adenocarcinoma Cells (HeLa Line)
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FRAPFluorescence
recovery after photo-bleaching
Study diffusion and movement of biological molecules◦ fluid mosaic model of
the cell membrane◦ study molecules in
the cytosol, nucleus, etc
Time
Fluorescence
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Fluorescence-Activated Cell Sorting
Rapid sortingSorts cells one-by-one
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MicroarraysDNA (Gene)
microarrays◦ Gene expression
profiling (using fluorescent labeled mRNA)
◦ SNP detectionProtein
microarrays◦ Antibody analysis◦ Protein
interactions
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Reporter GenesAttached to genes of interestChosen by the characteristics
they confer to the organism expressing them◦Easily identified / measured◦Selectable markers
Determine whether the gene of interest is being expressed
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Common uses of reporter genes
Gene expression assaysPromoter assaysTransformation / transfection
assaysTwo-hybrid screening
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So, what makes a good reporter gene?
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So, what makes a good reporter gene?Genes that confer easily
identifiable characteristics.◦Green Fluorescent Protein (GFP)
Jellyfish Causes cells to glow green under blue
light
◦Red Fluorescent Protein (dsRed) Coral
◦Luciferase Fireflys Catalyzes a reaction with luciferin,
producing light
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GFPAequorea victoria238 amino acidsRefined from WT over
the years◦ 1995; Mutation
dramatically improving the spectral characteristics of GFP
◦ 1995; F64L, allowing GFP use in mammalian cells
Variants◦ Superfolder GFP◦ Blue, Cyan, Yellow, Red,
Emerald, Apple……
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Fluorescent proteins and their uses
Fluorescent proteins derived from GFP and dsRed.
Colors: ◦ BFP◦ mTFP1◦ Emerald◦ Citrine◦ mOrange◦ mApple◦ mCherry◦ mGrape
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Florescent proteinsFluorescence microscopy
◦Florescent proteins not phototoxic, as are most florescent molecules
Determine when gene is expressed◦Exhibit morphological distinctions◦View biological processes (protein
folding, transport, etc)◦Expression of a florescent protein in
specific cells Optical detection of specific cells
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Two color male pig kidney epithelial cells undergoing mitosis
A culture of pig kidney cells
mCherry fused to human histone H2B
mEmerald fused to alpha-tubulin
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Use of GFP to identify specific cells
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GFP to identify cellular parts
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Expression of GFP to track specific cells
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Fluorescent proteins and their uses
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Fluorescent proteins and their uses
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Fluorescent proteins and their uses
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Fluorescent proteins and their uses
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Fluorescent proteins and their uses
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The End