Development
What causes cell differentiation?
Molecular Markers
Homeobox Genes
1. Transcription factors encoding the DNA binding homeodomain.
2. Expressed in temporal and spatial specific patterns during development.
3. Cell differentiation is disrupted when they are mutated.
Adult Spinal Cord
Neural Tube Development
How are the expression patterns for homeodomain proteins created?
Lbx1 Expression Pattern
Pax3•Lbx1
Project Goal
Identify the tissue specific enhancer that creates the Lbx1 expression pattern in the dorsal horn of the spinal cord.
Mouse Lbx1 Locus
Delivery of Reporter Constructs to Embryonic Cells
• Different experimental models can be used.
– Mouse common but difficult, expensive.
– Chick easy, inexpensive.
• Our method of choice: electroporation of chick embryos.
• Method is relatively new.
In Ovo Electroporation: GFPß-actin
EnhancerGFP
Instruments
Positive Control: ß-Actin GFP Expression Vector
Asymmetric Expression of GFP
In Ovo Electroporation: LacZCytomeglavirus
EnhancerLacZ
Positive Control: ß-Galactosidase Reporter Gene
Results
Conclusion
• Tissue specific enhancer does not exist within our search area.
• Can now start searching upstream and downstream.
Acknowledgements
Dr. Michael Gross
Dr. Kevin Ahern
Howard Hughes Medical Institute
Questions?