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Screening of Evolutionarily Conserved Enhancers in the Embryonic Spinal Cord Joe Knoedler, Amgen Scholars Program Gene Expression Laboratory at the Salk Institute Primary Investigator: Dr. Samuel Pfaff Introduction In eukaryotes, genes can be regulated by non-coding regions termed ‘enhancers’. Enhancers differ from conventional promoters in that they can be hundreds of thousands of base pairs away from the genes they regulate. The Pfaff lab’s primary focus is to determine the genetic programs governing the development of the circuitry of the spinal cord. Enhancers may play an important role in this event. To this end, my objective was to investigate the expression pattern of evolutionarily conserved enhancer regions (ECRs) in the chick spinal cord (Figure from Pfaff and Goulding 2005). Experimental Procedure Vectors were prepared by cloning ECRs from human genomic DNA using PCR. They were then ligated into backbones containing a CRErecombinase. The constructs were cultured in E. coli. Chick embryos were transformed with the construct and an RFP reporter plasmid via in ovo electroporation. Preliminary Investigation Collaborators in the Pennachio Lab (Lawrence Berkeley National Laboratory)identified ~1100 noncoding regions conserved in human, chick and mouse. They then made transgenic mice with the putative enhancer genes coupled to ßgalactosidase and used whole-mount stating to get a gross overview of expression patterns. 281 of these were active in the neural tube (Figures courtesy of William Alaynick). Results Section of neural tube of transformed chicken embryo. Cells that have taken up the RFP construct fluoresce red; cells that have taken up both and activate enhancer region express a CRE-recombinase that turns RFP into GFP. Thus, this enhancer may be involved in differentiation of green cells. Acknowledgements Enhancers in Development Enhancers can regulate development by regionally targeting gene expression. Left, a construct with whole Hb9 promoter targets expression to ventral neural tube; right, truncation of enhancer drives nonspecific GFP expression across dorso-ventral axis (Figure from Lee et al 2004) Special thanks to: Magda Gramada, director of UCSD Amgen Scholars program, Will Alaynick, my postdoctoral mentor, Sam Pfaff, my PI, and the Amgen Foundation for funding my internship. References Goulding, M. and Pfaff, S. L. (2005). Development of Circuits that Generate Simple Rhythmic Behaviors in Vertebrates. Current Opinion in Neurobiology 15, 14-20. Lee, S., Jurata, L. W., Funahashi, J., Ruiz, E. C. and Pfaff, S. L. (2004). Analysis of Embryonic Motoneuron Gene Regulation: Derepression of General Activators Function in Concert with Enhancer Factors. Development 131, 3295-3306.