In this experimental module you will modify the gene for EGFP (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event. We will then choose a few variables to test on the final day of the experiment.
Note: PDF reprints are provided below within the context of fair use. Please obtain copies from the publisher if appropriate.
Pathways for mitotic homologous recombination in mammalian cells Mutation Research 27 November 2003 Thomas Helleday URL
Homologous recombination as a mechanism of carcinogenesis Biochim Biophys Acta 21 March 2001 Bishop AJ and Schiestl RH URL
Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death EMBO J 15 January 1998 E Sonoda, M S Sasaki, J M Buerstedde, O Bezzubova, A Shinohara, H Ogawa, M Takata, Y Yamaguchi-Iwai, and S Takeda PDF reprint