gfp: lighting up life · my companions and i then witnessed a curious spectacle. . .the nautilus i...
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My companions and I then witnessed a curious spectacle. . .The Nautilus floated in the midst of. . . truly living light. . . an infinite
agglomeration of
colored. . . globules of diaphanous jelly. . .
Twenty Thousand Leagues Under the Sea –
Jules Verne
Now it is such a bizarrely improbable coincidence that anything so mind- bogglingly useful could have evolved purely by chance that some thinkers have chosen to see it as a final and clinching proof of the nonexistence of God.
The Hitchhiker’s Guide to the Galaxy – Douglas Adams
GFP: Lighting Up Life
You can observe a lot by watching.Yogi Berra
Wilhelm Röntgen Santiago Ramón y Cajal Camillo Golgi
Ultramicroscope
RichardZsigmondy
Chemistry, 1925
Phase Contrast Microscope
FritsZernike
Physics, 1953
Large-
ArrayRadio
Telescopes
MartinRyle
Physics, 1974
ElectronMicroscope
ErnstRuska
Physics, 1986
X-ray Crystallography
Physics, 1915
LawrenceBragg
WilliamBragg
Nuclear Magnetic Resonance
Physics, 1952
E. M.Purcell
FelixBloch
Scanning Tunneling Microscope
Physics, 1986
HeinrichRohrer
GerdBinnig
Computer Assisted Tomography
Physiology or Medicine, 1979
Godfrey Hounsfield
AllanCormack
Magnetic Resonance Imaging
Physiology or Medicine, 2003
PeterMansfield
PaulLauterbur
Sydney BrennerJosé
Zadunaisky Bob Perlman
Sydney Brenner Bob Horvitz John Sulston
Caenorhabditis elegans
β-galactosidase Activity
mec-9
Antibody Staining
MEC-7
Hongping Du
Aequorin + Ca++
+ GFP light
Aequorin + Ca++ light
Paul Brehm Osamu ShimomuraAequorea victoria
Douglas Prasher
The GFP Fluorophore
Tyr66
Phe64
Ser65
. . .
. . . NH-
1
TACACACGAA TAAAAGATAA CAAAGATGAG TAAAGGAGAA GAACTTTTCA CTGGAGTTGT61 CCCAATTCTT GTTGAATTAG ATGGTGATGT TAATGGGCAC AAATTTTCTG
TCAGTGGAGA121 GGGTGAAGGT GATGCAACAT ACGGAAAACT TACCCTTAAA TTTATTTGCA CTACTGGAAA181 ACTACCTGTT CCATGGCCAA CACTTGTCAC TACTTTCTCT TATGGTGTTC AATGCTTTTC241 AAGATACCCA GATCATATGA AACAGCATGA CTTTTTCAAG AGTGCCATGC CCGAAGGTTA361 CAAGTTTGAA GGTGATACCC TTGTTAATAG AATCGAGTTA AAAGGTATTG ATTTTAAAGA421 AGATGGAAAC ATTCTTGGAC ACAAATTGGA ATACAACTAT AACTCACACA ATGTATACAT481 CATGGCAGAC AAACAAAAGA ATGGAATCAA AGTTAACTTC AAAATTAGAC ACAACATTGA541 AGATGGAAGC GTTCAACTAG CAGACCATTA TCAACAAAAT ACTCCAATTG GCGATGGCCC601 TGTCCTTTTA CCAGACAACC ATTACCTGTC CACACAATCT GCCCTTTCGA AAGATCCCAA661 CGAAAAGAGA GACCACATGG TCCTTCTTGA GTTTGTAACA GCTGCTGGGA TTACACATGG721 CATGGATGAA CTATACAAAT AAATGTCCAG ACTTCCAATT GACACTAAAG TGTCCGAACA781 ATTACTAAAA TCTCAGGGTT CCTGGTTAAA TTCAGGCTGA GATATTATTT ATATATTTAT841 AGATTCATTA AAATTGTATG AATAATTTAT TGATGTTATT GATAGAGGTT ATTTTCTTAT
λGFP10
EcoRI EcoRI
Ghia
Euskirchen
Yuan Tu
Bill Ward
Green Fluorescent Protein: A New Markerfor Gene Expression
The Aequorea victoria Green Fluorescent Protein Needs No Exogenously-Added Component to Produce a Fluorescent
Product in Prokaryotic and Eukaryotic Cells
Martin Chalfie, Yuan Tu, Ghia
Euskirchen, William W. Ward, Douglas C. Prasher
Science 263: 802-805, 1994
Green Fluorescent Protein as a Markerfor Gene Expression
Tulle Hazelrigg
Sarah Chalfie
Shengxian
Wang and Tulle Hazelrigg
Shengxian
Wang
Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis
Nature 369: 400-403, 1994Tulle Hazelrigg
Advantages of GFP as a Biological Marker
1.
Heritable
2.
Relatively Non-invasive
3.
Small and Monomeric
4.
Visible in Living Tissues
Improving GFP
Tyr66
Emission
Ser65 ExcitationRoger Tsien
Papers Using Green Fluorescent Protein
The First Human GFP Transgenic?
Ang
Lee
PVM
AVMPLM
ALMPmec-17 gfp
Gene Expression
Yun
Zhang
FLP FLP FLP
EGL-44::YFP EGL-46::CFP MERGED
Co-expression
Ji
Wu
Pmec-4 cfp
mec-4::yfpDattananda
Chelur
Protein Localization
MEC-1 MEC-9 MEC-5
MEC-7MEC-12
MEC-4 MEC-10
MEC-6
MEC-2β-tubulin
α-tubulin
collagenKunitz/EGF proteins
DEG/ENaC
PHB-domain proteins
paraoxonase
UNC-24
unc-51
mec-7
Wild Type
Mutant Screens and Characterizstion
Hongping
Du
gfprab-3
Mike Nonet
Visualizing Synapses
wild type
mec-15
Alex Bounoutas
Pmec-7
Yun
Zhang
Cell Isolation
Irini
Topalidou
Touch neuronsFLP neurons
The Problem with C. elegans Electrophysiology
Bob O’Hagan
Cell-specific Electrophysiology
MEC-4 MEC-10
MEC-6
MEC-2
Miriam
Goodman
Interaction ofLeucine
Zippers
Reconstitution of Functional GFP
recGFP
NZGFP
NZ CZ
linker
NGFP CGFP
linker
CZGFPFrom Promoter A From Promoter B
A B
Non-covalent Reconstitution of GFP
Lynne Regan
Indraneel
Ghosh, Andrew D. Hamilton, and Lynne Regan (2000) Antiparallel
Leucine
Zipper-Directed Protein Reassembly:
Application to the Green Fluorescent Protein. J. Am. Chem. Soc. 122: 5658–5659.
Refining Cell Labeling
mec-3 egl-44
FLP
Intestine
Pmec-3 nzgfp & Pegl-44 czgfp
PVDPLM
AVMALM
PVM
Hypodermis
HSNOther
neurons
Shifang
Zhang Chuck Ma
L1 L4 Adult
Control
Colchicine
Mutagenesis
Colchicine
Alex Bounoutas
Chuck Ma
Leslie Emtage
praja gfpPmec-18
MEC-3 MEC-7 + MEC-12 microtubulesHomeodomainTranscription
Factor
β-tubulin α-tubulin
Madeline Friedlen
Vivian Chalfie Eli Chalfie
Postdoctoral Scientists
Anahita
AmiriGuy Caldwell Lucinda CarnellAnne ChambersDattananda
ChelurBrian CoblitzMonica Driscoll Chip Ferguson Nikolaos
George Miriam GoodmanJang Hee
HahnChris Li Charles Ma Chris MartinShohei
MitaniErich Schwarz Anne TokerIrini
TopalidouMillet Treinin
Bill Walthall Jeff WayEve Wolinsky
Graduate Students
Alex BounoutasAndrea CalixtoXiaoyin
ChenYushu
ChenHongping
DuAnne DugganLesley EmtageGlen ErnstromGhia
EuskirchenJaime García-
AñoverosGuoqiang
GuMingxia
HuangSiavash
KarimzadeganCharles Keller
John KratzJoy LiangShujun
LuoCathy Savage Jim TaubJi
Wu Yi-Chun Wu Ding XueShifang
Zhang Yun
Zhang
Undergraduates
Macy Au Paul AuwaeterPeggy BrickmanKate BraumanJohn ByunAlex ChangVictor ChangIris ChinBradley CollinsMichael Dreyfuss
AngelaGeorgopoulos
Rachel GoldsteinJudith GreenPeter HomerRafaz
HoqueJames HudspethShari JawetzPaul JosephsonEric KanterLexy
KovachJoe LauSam LeeLorraine
LothringerDavid MeshoulamJeremy MindichMariya
RosenblitJulie RosenthalAaron SchefflerShai
ShahamJay SrinivasanLeslie Vosshall
Molly Weiner Chris William
Technicians
Margaret BarnesElke
BergholzLei Chen Ellen DeanNora HomMaud KinnellConnie MauokaEvelyn ReillyYuan TuYingzi
XueAndrea Yao
FacultyVisitors
Christian NeriJonathan
Rothblatt
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