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Identification and Comparison of Midline Cis- Regulatory Elements

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

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Identification and Comparison of Midline Cis- Regulatory Elements

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  1. Identification and Comparison of Midline Cis-Regulatory Elements

  2. 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

  3. 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

  4. Sim 2.8 pE Sim (Sandmann) sli380 rho E-Ss Rst F6d Kr PP3.0Hz Kr StH0.6Hz btl-23 tl950 Known Midline Enhancers

  5. Candidate Midline Enhancer Sources(midline database search) Midline Glia Midline Primordia 13 19 69

  6. St. 11 Midline Genes bnb ab argos bib cdi CG13333 cenB1A CG31145 CG8291 CG32594 CG3409 CG7224 ct dve glec CG9634 hbs HGTX oc mfas sty Tkr vvl sog Sema-1b btl rho rst sim Tl

  7. mfas oc rho sim sty wrapper alan-shepard slit GH22170 Midline/Glial Expressing Genes • argos • cdi • CG13333 • CG31145 • CG8291 • CG9634 • cut • dve • glec • hbs

  8. CG7224 cenB1A (Sema-1B) bib bnb CG3409 CG32594 HGTX sog ab Tkr ct vvl Midline Primordium Genes

  9. Enhancer Cloning Pipeline AttR1 AttR2 AttL1 AttL2 fC31 AttB Reporter Reporter Vectors Entry Vector

  10. 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)

  11. Rare MCS pENTR vector NgoMIV/ NaeI SbfI PmeI SpeI PstI NcoI AscI FseI NotI/EagI DraI AttL1 AttL2 pENTR/D-Topo pGEM-T sites: NcoI, NotI (both sides), NsiI, PstI, SpeI pCRII sites: NotI, NsiI (both sides), SpeI, XbaI Compatible ends: NotI/EagI SbfI/PstI->(NsiI) NgoMIV->(AgeI)/(XmaI) NcoI->(BspHI)/(PciI) SpeI->(AvrII)/(NheI)/(XbaI) AscI->(MluI)/(BssHII)

  12. Reporter Vector Issues • pBPGw has GAL4 • pBPGw doesn’t have a promoter • pBPGw is named pBPGw

  13. pMintGate pBPJPhGFPw pBPJPhGFPw

  14. Test Case:Sim2.8 pE fC31 AttB fC31 AttB fC31 AttB GAL4 GFP.nls DsRed.nls MintGate CinnamonGate pBPGw

  15. 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)

  16. GAL4-UAS Cell-specific gene rescue RNAi Cell labeling Enhancer Dissection Common Activation? Common Repression? Evolutionary Conservation? Applications of Enhancers

  17. 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

  18. ChIP-Seq FACS + Fix chromatin, IP with SIM Solexa Sequence

  19. FAIRE

  20. FAIRE FACS + Fix chromatin, Phenol Extract Free DNA Label DNA, Custom Microarray

  21. FAIRE Midline cells Midline Subtracted geneA geneA geneB geneB geneC geneC A B C D E F G H I J K L A B C D E F G H I J K L

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