GFP tagged Zebrafish a model for heat shock protein studies

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GFP tagged Zebrafish - a model for heat shock
protein studies
Nima Maria George
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Nobel prize in Chemistry-2008

• Osamu Shimomura, Boston University Medical School
  - GFP identification
• Martin Chalfie, Columbia University - GFP as a
  genetic tag
• Roger Y. Tsien, University of California -how
  GFP fluoresces "for the discovery and
  development of the green fluorescent protein,
  GFP".

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• Shimomura went to Friday Harbor, Washington,
  to collect the squeezate and to extract from it
  the substance responsible for its luminescence.
  In order to do his research, he collected over a
  million Aequorea specimens, cut off the rings,
  and produced squeezate..

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Martin Chalfie-expressed GFP in E.coli and in
Cenorabditis elegans Green Fluorescent protein
as a marker for gene expression-1994
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Roger Tsien Made a single point mutation that can
change the spectral and fluorescent
characteristics of GFP
GFP chromophore formation requires oxygen  
Made variants of GFP can shine more brightly and
show different colors, such as yellow, cyan, and
blue.
Cloning of the cDNA of GFP in 1992 by Douglas
Parsher
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About GFP

• From Jelly fish, Aequorea victoria
• Produced in the photo organs
• Luminescent protein Aequorin interacts with Ca2
  and produces blue light
• Blue light is captured by GFP and gives off green
  light
• Emission peak is at 509 nm ,lower green portion
  of the visible spectrum.

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Main attractions

• Small protein of 238 amino acids
• Active as a monomer
• Can be excited by uv or blue light
• Wont photobleach easily
• No cofactors needed

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Applications

• Reporter for gene expression
• Marker to study cell lineage during development
• Tag to localize proteins in living cells
• Can follow the protein movement or study the
  dynamics of the subcellular compartments to
  which it is targeted

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Structure
Primary sequence
After cyclization
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Structure
Beta barrel structure One ß-sheet with alpha
helix(s) containing the chromophore running
through the center. Cyclization reactions in the
tripeptide Ser65Tyr66Gly67 that lead
to chromophore formation.
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Heat Shock Proteins(Hsps)

• Conserved group of proteins produced upon
  environmental stress
• Protects the vital cellular functions
• Helps in protein folding and prevents aggregation
• Carry old proteins to the cell's "recycling bin"
  (proteasome) 
•  Form a part of cell's own repair system, called
  the "cellular stress response" or the "heat-shock
  response"

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Why Zebra fish for hsp studies?

• Large fecundity
• Externally developing and transparent embryos
• Rapid development
• Ease of manipulation
• Generation of transgenic lines
• Hsps in transgenic fish as a model of toxicology
  studies

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• A 2.2 kb 5upstream region and hsp27 gene is
  amplified from zebrafish genomic clone by PCR
• Plasmid pEGF-1 with GFP transgene was
  microinjected into zebrafish embryos at 1-2
  celled stage
• GFP florescence hour post fertilization is
  analyzed
• Heat shock treatment(37degrees for 1-2 hr) from
  4hpf to 8dpf

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• Heavy metal treatment of 72hpf transgenic larvae-
  Sodium arsenate and Cadmium chloride
• Heavy metal treatment for 48 hrs
• Comparison of endogenous and GFP gene expression
  of hsp 27 is done

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Whole mount insitu hybridization of Hsp mRNA
using antisense RNA probe for endogenous
expression Heat shock causes clear increase of
the expression in different stages of
growth
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• Expression starts 11hpf
• Highest expression at 7dpf

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Higher expression in muscles, eyes, skin and
heart as compared to the untreated control
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• Expression started 72 hpf
• GFP expression in skeletal muscles after 24hpf
  and 48hpf treatment

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• No induction upon Cadmium treatment
• Increased expression of GFP upon sodium arsenate
  treatment
• Heart, Skeletal and Cranial muscles showed
  increased expression

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Conclusion

• hsp27 gfp transgenic fish is responsive to heat
  shock
• Highest expression in skeletal muscles
• Recapitulates the expression pattern and
  inducibility of endogenous hsp27 RNAs
• Heavy metal induction of hsp27 by Arsenic

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Thank you