Identification and Comparison of Midline Cis- Regulatory Elements
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Transcript of Identification and Comparison of Midline Cis- Regulatory Elements
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Identification and Comparison of Midline Cis-Regulatory Elements
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Does common expression indicate common regulation?
A
B
C
DC D
A B
A
B C D
C DA B
w x y z
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Project Goals
• Identify Large Set of Midline Primordium Enhancers
• Compare enhancers for shared motifs
• Experimentally confirm required motifs (activators/repressors)
• Contribute to knowledge of Midline Development
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Known Midline Enhancers
• Sim 2.8 pE
• Sim (Sandmann)
• sli380
• rho E-Ss
• Rst F6d
• Kr PP3.0Hz
• Kr StH0.6Hz
• btl-23
• tl950
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Candidate Midline Enhancer Sources(midline database search)
Midline Primordia
Midline Glia
1913 69
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CG13333
St. 11 Midline Genesargosab bib bnb
btl
cdi cenB1A CG31145
CG32594 CG3409 CG7224 CG8291
ct dve glec
hbs HGTX mfas oc
rhoSema-1b
sog sty vvlTkr
CG9634
rst sim Tl
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Midline/Glial Expressing Genes
• mfas• oc• rho• sim• sty• wrapper• alan-shepard• slit• GH22170
• argos• cdi• CG13333• CG31145• CG8291• CG9634• cut• dve• glec• hbs
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Midline Primordium Genes
• CG7224
• cenB1A
• (Sema-1B)
• bib
• bnb
• CG3409
• CG32594
• HGTX
• sog
• ab
• Tkr
• ct
• vvl
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AttL2
Enhancer Cloning Pipeline
Entry VectorReporter Vectors
AttL1AttR2AttR1
Reporter
C31
At
tB
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Entry Vector Issues
• pENTR/D-TOPO does not like big fragments
• Enhancer hunts do like big fragments
• Solutions:– Use Restriction/Ligation into pENTR/D-TOPO– Use pCR8/GW/TOPO vector (TA cloning)
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NotI/EagI PstISbfI
NgoMIV/NaeIFseI NcoI DraI
PmeISpeI AscI
Compatible ends:
NotI/EagI SbfI/PstI->(NsiI) NgoMIV->(AgeI)/(XmaI)
NcoI->(BspHI)/(PciI) SpeI->(AvrII)/(NheI)/(XbaI) AscI->(MluI)/(BssHII)
pGEM-T sites: NcoI, NotI (both sides), NsiI, PstI, SpeI
pCRII sites: NotI, NsiI (both sides), SpeI, XbaI
Rare MCS pENTR vector
AttL2AttL1
pENTR/D-Topo
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Reporter Vector Issues
• pBPGw has GAL4
• pBPGw doesn’t have a promoter
• pBPGw is named pBPGw
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pMintGate
mintGate9679 bp
pBR322 origin
GFP-NLS stop
SV40 3'UTR
EGFP 5'UTR
ChlorR
ccdB
EGFP
tra NLS
mini-white
AmpR
AttR
AttR
phiC31 AttB
Apa I (4225)
Bgl II (1804) Kpn I (1847)
Nde I (7564)
Sac I (5688)
Xho I (1869)Bam HI (1863)
Eco RI (1826)
Spe I (3165)
Asc I (1836)
Asc I (3427)
Asc I (7554)
Xba I (1812)
Xba I (2934)
Xba I (3171)
pBPJPhGFPw
pBPJPhGFPw
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Test Case:Sim2.8 pE
MintGate
GF
P.nls
C31
At
tB
CinnamonGate
DsR
ed.nlsC
31
AttB
pBPGw
GA
L4
C31
At
tB
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Preliminary results
• Cloned Sim2.8 pE into pMintGate, pCinnamonGate, pBPGw
• One Insertion isolated for Sim2.8 pMintGate (so far)
(10/29: 2 lines CinnamonGate, 1 line pBPGw)
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Applications of Enhancers
• GAL4-UAS– Cell-specific gene
rescue– RNAi
• Cell labeling
• Enhancer Dissection– Common Activation?– Common Repression?– Evolutionary
Conservation?
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Future Experiments
• Clone/Test Putative Enhancer Fragments– Best case: 30+ midline enhancers with similar
expression– compare bioinformatically– Identify required motifs
• Replace current FPs with newer/better FPs– mCherry, mPlum, Cerulean, CFP, etc– Live, in vivo comparisons of multiple midline enhancers
• Genome-wide midline enhancer tests– Chip-Seq– FAIRE
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ChIP-Seq
+
Fix chromatin, IP with SIM
FACS
Solexa Sequence
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FAIRE
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FAIRE
+
Fix chromatin, Phenol ExtractFree DNA
FACS
Label DNA,Custom Microarray
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geneA
geneB
geneC
geneA
geneB
geneC
Midline cells Midline Subtracted
ABCDEFGHIJKL
ABCDEFGHIJKL
FAIRE