Fluorescent Protein Trapping in Drosophila melanogaster
Open Access
- Author:
- Foltz, Anna
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Claire Madison Thomas, Thesis Supervisor
Song Tan, Thesis Honors Advisor - Keywords:
- Drosophila melanogaster
Gene Trap
Fluorescent Protein
Molecular Cloning
Larval Screening - Abstract:
- Drosophila melanogaster is a popular model organism with tractable genetics. To track protein levels and localization in vivo, gene traps are popular tools used to tether a fluorescent protein to a protein of interest in the fly genome. Historically, Green Fluorescent Protein (GFP) has been used for this purpose. In Dr. Claire Thomas’ 200-level Biology course Biol230M ‘The Biology of Molecules and Cells’, gene trapping has been used for several years to bring a research exercise into the laboratory classroom. This class used the existing Wee-P transposon, a GFP based gene trap. When the Wee-P transposon lands within introns at random across fly chromosomes, the Wee-P exon can be spliced to adjacent exons through splice sites located on either side of the protein coding sequence, generating a fluorescent protein fusion. Several years of experience in the lab has shown that the undergraduates consistently find it difficult to distinguish many GFP fusion events from the green-autofluorescence found in many Drosophila tissues. In this thesis I attempt to avoid this setback by making a new gene trapping transposon, based on the red fluorescent protein tdTomato. Reengineering this system also offers the opportunity to optimize several other aspects of the system. As well as improving trap detection with tdTomato, I also tried to optimize the trap identification process by designing better inverse PCR primers and more convenient restriction endonuclease sites to maximize the probability of success when using the iPCR gene identification strategy. Finally, the new design relocates the P[w+] ammunition construct’s selectable marker outside of the transposon to obviate the need to remove it after successfully trapping, simplifying the creation of a stable stock. We intend to make the new trap lines freely available to the Drosophila community, and I hope that some of the class-generated stocks will eventually prove useful to other fly labs.