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Hox Gene Expression Pattern

Hox Gene Expression Pattern. B13. How is the expression of Hox genes regulated?. Local regulatory elements Long range regulatory elements. Classical Methods. Reporter. Reporter Technique. Make Transgenic Mice Analysis Expression Pattern. Sharing and selectivity. wt. Hoxb5. Sharing.

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Hox Gene Expression Pattern

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  1. Hox Gene Expression Pattern B13

  2. How is the expression of Hox genes regulated? • Local regulatory elements • Long range regulatory elements

  3. Classical Methods Reporter Reporter Technique Make Transgenic Mice Analysis Expression Pattern

  4. Sharing and selectivity wt Hoxb5 Sharing Selectivity Hoxb4 wt Sharpe J. et.al; EMBO J; 17(6),1998

  5. Local Regulatory Elements

  6. How is the expression of Hox genes regulated? • Local regulatory elements Larger number; Sharing and selectivity are common. • Long range regulatory elements Largely unknown Problem: the size limitation of molecular cloning technique.

  7. Bacterial Artificial Chromosomes (BACs) • E.Coli F Factor Based Cloning System • 1-2 Copies Per Cell • Allow up to 350Kb DNA Inserts • Easy to Purify (Standard Plasmid Isolation Methods) • Many Libraries Avaliable

  8. Tim’s Experiments LacZ Transposon neural, meso.

  9. Purposes of this project: Aim I: Further optimize the Bac system for the study of long range regulation Aim II: Study the long range effect of the local elements between HoxB4 and HoxB5 Aim III: Study the 3’ boundary structure of HoxB complex

  10. Aim I: Bac System • Transposon Mediated Enhancer Trapping • Homologous Recombination

  11. Transposon Transposon Mediated Enhancer Trapping

  12. Homologous Recombination Selection Marker Modification Cassette 50nt 50nt Homologous Recombination DH10B Strain w/ Defective Pro Phage Bac of Interested Direct Selection A B Selection Marker Modification Cassette Vector A B Bac of Interested Transformation Vector Selection Marker A Modification Cassette B CmR Selection Bac of Interested Pick Up a Single Clone and Culture PCR 42oC 15 min Expression of Gam, Exo and Bet Marker Selection

  13. +- Selection Positive - Negative Selection A B Negative Marker Positive Marker Vector A B Bac of Interested Positive Selection Vector Positive Marker Negative Marker A B Bac of Interested Modification Cassette A B Negative Selection Vector Modification Cassette A B Bac of Interested SacB AmpR GFP

  14. Deletion Deletion and Subcloning • Deletion B Selection Marker A Vector B A Bac of Interested 2. Subcloning AmpR ori A B Vector B A Bac of Interested

  15. b2,b1 b13-b7 YFP RFP GFP BFP Aim II: Long Range Effect of HoxB4-HoxB5 Local Elements Experiment Design Do the expression patterns change?

  16. Aim III: Boundary Structure Experiment Design I Do the expression patterns change?

  17. GFP GFP Boundary Structure Experiment Design II Oct-4 Enhancer 5’ 3’ Other HoxB genes HoxB1 Oct-4 Enhancer 5’ 3’ HoxB BACs Neural, Mesoderm Gut When does HoxB1 GFP express?

  18. Thanks Acknowledgments: Robb Krumlauf Paco Cambronero Paul A. Tranior Kristen Corriea Debra Ellies Linheng Li’s Lab Takayoshi Inoue Heather Marshall Neal G. Copeland (NCI) Kieran Pemberton Stefan Tuempel Leanne Wiedemann Timothy Jinks

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