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Combinatorial Marking of Cells and Organelles with Split Fluorescent Proteins. Shifang Zhang, Charles Ma, & Martin Chalfie Department of Biological Sciences, Columbia University. OUTLINE. Background The principle of split GFP The test of split GFP in C.elegans - PowerPoint PPT Presentation
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Combinatorial Marking of Cells and Organelles with Split Fluorescent
Proteins
Shifang Zhang, Charles Ma, & Martin Chalfie Department of Biological Sciences, Columbia University
OUTLINEOUTLINE BackgroundBackground The principle of split GFPThe principle of split GFP The test of split GFP in The test of split GFP in C.elegansC.elegans
1.1. The split GFP can be expressed in The split GFP can be expressed in C.elegansC.elegans with P with Pmec-18 mec-18 2. Split GFP was not promoter or tissue dependent3. Reconstitution was not restricted to split GFP
The use of split GFPThe use of split GFP1. Split GFP can identify cells that coexpress different promoters2. Split GFP can be used demonstrate changes in gene expression3. Split GFP can also be used to identify cells expressing a
particular gene4. Split GFP can be used to label cell constituents in a restricted set
of cells
BackgroundBackground
Advantage: encoded Advantage: encoded and expressed in and expressed in livingliving cells and cells and organismsorganisms
Limited: individual Limited: individual cell types cannot be cell types cannot be labelled using single labelled using single regulatory elementregulatory element
The split GFPThe split GFP
2000, 2000, Indraneel Ghosh
NZ: ALKKELQANKKELAQLKWELQALKKELAQCZ: EQLEKKLQALEKKLAQLEWKNQALEKKLAQ
NZGFP=NGFP + 6 amino acid linker + NZ
CZGFP=CZ + 4 amino acid linker + CGFP
The test of split GFP in The test of split GFP in C.elegansC.elegans
Split GFP can be expressed in Split GFP can be expressed in C.elegansC.elegans
PPmec-18 mec-18 :: only expressed in the six touch only expressed in the six touch receptor neuronsreceptor neurons
PPmec-18 nzgfp mec-18 nzgfp + P+ Pmec-18 mec-18
czgfp czgfp
When both parts of split GFP were expressed
If only one part of the split GFP was expressed, If only one part of the split GFP was expressed, there is no fluorescence can be observed.there is no fluorescence can be observed.
The fluorescence did not result from DNA rearrangement during transformation
PPmec-18 nzgfp mec-18 nzgfp + + czgfpczgfp
fluorescencfluorescencee
The absence of CZ prevented the production of fluorescence
PPhsp16.2 hsp16.2 : heat shock promoter: heat shock promoter
Split GFP was not promoter or tissue dependent
PPhsp16.2 nzgfp hsp16.2 nzgfp ++ PPhsp16.2 czgfphsp16.2 czgfp
The split GFP has a shorter half-life than GFP
Punc-4nzgfp& Punc-4czgfp
Punc-4gfp
<2 hr
20 hr
40 hr
PPunc-4 unc-4 : expressed in : expressed in four types of motor four types of motor neurons (SAB, VA, DA, neurons (SAB, VA, DA, and VC)and VC)
Masha et al., 2003
Reconstitution was not restricted to split GFP
CFP Filter SetYFP Filter Set
NZYFP+CZCFP
NZGFP+CZCFP
NZCFP+CZCFP
NZYFP+CZYFP
NZGFP+CZGFP
The use of split GFPThe use of split GFP
Punc-24 nzgfp+ Pmec-2 czgfpPunc-24 : expressed in C.elegans touch recetor neurons
and in many cells in the ventral cord
Pmec-2 : expressed in the six touch receptor neurons
Split GFP can identify cells that coexpress different promoters
Punc-24gfp
Punc-24nzgfp& Pmec-2czgfp
Pmec-3nzgfp& Pegl-44czgfp
only the two FLP neurons
fluoresced
Pmec-3 Pegl-44
touch receptors
PVD neurons
FLPneurons
HSN neurons
a member of the transcription enhancer factor family
Split GFP can be used demonstrate changes in gene expression
1999, Winnier
Punc-4 nzgfp + Pacr-5 czgfp
Split GFP can also be used to identify cells expressing a particular gene
Which motor neurons of the ventral cord
VD and DD motor neurons
VA, DA and possibly VC neurons
VB and DB motor neurons
In the excitatory motor neurons but not the inhibitory motor neurons
With the color coding provided by the different NZ fluorescent proteins, relatively few (perhaps less than thirty) strains could be used to characterize gene expression in all of the 302 C. elegans neurons (118 classes)
Px NZCFP X=Pb NZCFP
Pc NZYFP
Pa NZGFP
A possible extension
The apparent short half life of split GFP raises an important caution about negative results
HSN neurons egl-44 : in the embryocat-1 : in the late larval
HSN fluorescence was weak and rarely seen when split GFP was generated from these promoters
Punc-4nzgfp& Psto-6czgfp
Punc-4nzgfp& Punc-4czgfp
Split GFP can be used to label cell constituents in a restricted set of cells
synaptobrevin::GFP (SNB-1::GFP) protein fusion localizes to presynaptic vesicles
Cell bodies and processes of the B motor neurons in ventral cord
Presynaptic regions
Nuclei
The usage of GFP should be aided by the use of split fluorescent proteins
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