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?