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Insulator Studies in Drosophila melanogaster : 

Insulator Studies in Drosophila melanogaster Matt Borok Drewell Lab 2/20/09

Outline : 

Outline What is an insulator? Current projects and progress

How do enhancers upregulate gene expression? : 

How do enhancers upregulate gene expression? Transcription factor recruitment Interaction with gene promoters Insulator

What are enhancer-blocking insulators? : 

What are enhancer-blocking insulators? Regions of DNA that block interactions between enhancer and promoter Only block interactions when between the enhancer and the promoter Create regulatory domains

Direct Contact : 

Direct Contact Proteins bind to insulator sequence and form chromatin loops Interactions favored between elements in the same loop

The Gypsy Loop : 

The Gypsy Loop

Slide 7: 

Reporter construct is on P-Element P-Element is transposon or “jumping gene” Will integrate randomly into genome with help of transposase Creating Transgenic Flies Cross grown-up flies with white-eyed virgin flies Inject into embryo P-element with gene for red eyes Plasmid encoding transposase Screen for red eyed transgenic flies

FlyC31: A better method for transgenesis : 

FlyC31: A better method for transgenesis Site-specific insertion into fly genome Only one line needed attB vector Flies attP site phiC31 integrase

Site-specific integration : 

Site-specific integration

W-5-Z-attB : 

W-5-Z-attB

Slide 11: 

In situ results

The Bithorax Complex : 

The Bithorax Complex

New Fabs : 

New Fabs dCTCF predicted to bind many regulatory region boundaries in BX-C Responsible for insulator activity? Insulator activity at uncharacterized boundaries?

dCTCF-associated boundaries : 

dCTCF-associated boundaries

Goals : 

Goals Clone putative dCTCF-dependent insulators Test insulator activity in transgenic flies using placZattB vector Progress Fab-1,2,6 in W-5-Z-attB Fab-9 in pGEmT-Easy

Plasmid construction : 

Plasmid construction

Questions? : 

Questions?

Acknowledgements : 

Acknowledgements Johannes Bischof Drewell Lab HMC Biology Department

Synthetic CTCF insulator : 

Synthetic CTCF insulator dCTCF recently identified in flies Localizes to many boundaries of the BX-C Consensus sequence identified What is the minimal requirement for a CTCF-dependent insulator?

Consensus CTCF binding sites : 

Consensus CTCF binding sites D. mel Vertebrate

Goals : 

Goals Create synthetic consensus sequences 1 D. mel 1 vertebrate 1 D. mel + 1 vertebrate Spacing? Test insulator activity in transgenic flies using placZattB vector Progress

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