identification and comparison of midline cis- regulatory elements
DESCRIPTION
Identification and Comparison of Midline Cis- Regulatory Elements. A. B. C. A. B. D. C. D. Does common expression indicate common regulation?. A. C. D. B. w. x. y. z. A. B. C. D. Project Goals. Identify Large Set of Midline Primordium Enhancers - PowerPoint PPT PresentationTRANSCRIPT
Identification and Comparison of Midline Cis-Regulatory Elements
Does common expression indicate common regulation?
A
B
C
DC D
A B
A
B C D
C DA B
w x y z
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
Known Midline Enhancers
• Sim 2.8 pE
• Sim (Sandmann)
• sli380
• rho E-Ss
• Rst F6d
• Kr PP3.0Hz
• Kr StH0.6Hz
• btl-23
• tl950
Candidate Midline Enhancer Sources(midline database search)
Midline Primordia
Midline Glia
1913 69
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
Midline/Glial Expressing Genes
• mfas• oc• rho• sim• sty• wrapper• alan-shepard• slit• GH22170
• argos• cdi• CG13333• CG31145• CG8291• CG9634• cut• dve• glec• hbs
Midline Primordium Genes
• CG7224
• cenB1A
• (Sema-1B)
• bib
• bnb
• CG3409
• CG32594
• HGTX
• sog
• ab
• Tkr
• ct
• vvl
AttL2
Enhancer Cloning Pipeline
Entry VectorReporter Vectors
AttL1AttR2AttR1
Reporter
C31
At
tB
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)
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
Reporter Vector Issues
• pBPGw has GAL4
• pBPGw doesn’t have a promoter
• pBPGw is named pBPGw
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
Test Case:Sim2.8 pE
MintGate
GF
P.nls
C31
At
tB
CinnamonGate
DsR
ed.nlsC
31
AttB
pBPGw
GA
L4
C31
At
tB
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)
Applications of Enhancers
• GAL4-UAS– Cell-specific gene
rescue– RNAi
• Cell labeling
• Enhancer Dissection– Common Activation?– Common Repression?– Evolutionary
Conservation?
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
ChIP-Seq
+
Fix chromatin, IP with SIM
FACS
Solexa Sequence
FAIRE
FAIRE
+
Fix chromatin, Phenol ExtractFree DNA
FACS
Label DNA,Custom Microarray
geneA
geneB
geneC
geneA
geneB
geneC
Midline cells Midline Subtracted
ABCDEFGHIJKL
ABCDEFGHIJKL
FAIRE