Researcher(s)
- Raktim Basu, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Mona Batish, Department of Medical and Molecular Sciences, University of Delaware
Abstract
Single-molecule fluorescence in-situ hybridization (smFISH) is a fluorescence-based imaging technique that can identify the presence of tagged nucleotide sequences by use of selective complementary sequences known as probes. SmFISH can further be refined by use of a 2-step process, with an unlabeled branched primary probe designed to be complementary to the target sequence, and a labeled secondary probe that attaches to the branched primary probe enabling visualization of the target sequence. I used the 2 step smFISH to image different splice variants of the BOK gene. The BOK (Bcl-2 related ovarian killer) is a protein-coding gene known to induce apoptosis via a yet uncharacterized mechanism and is shown to have a role in regulating proliferation of various cancers. The splice variants of the BOK gene vary between cancers. Several exons, primarily exon 2, 4, 5 and 6, are shared across several splice variants that have important roles in cancers. Ability to distinctly image different splice variants will help in determining their distribution that can be correlated to their function in different cancers.
Using a previously established method of smFISH involving a primary and secondary probe, Caco-2 cells (cells isolated from the colon of a patient with adenocarcinoma) were cultured with DMEM media containing 20% FBS and 1% Pen-Strep. Glass coverslips were sterilized with 100% EtOH and 70% EtOH, and then coated with 0.01% gelatin to ensure the attachment of Caco-2 cells. Cells were grown on coverslips around 70-80% confluency and then they were fixed and permeabilized using 4% PFA and 70% ethanol, respectively..
The coverslips were then hybridized according to the smFISH protocol as previously described in Batish et. al.
Primary probes designed to bind to exon 2 and probes designed to bind to exons 4, 5, and 6 all showed clear and detectable spots of intensity during imaging. However, the procedure has not been fully optimized for Caco-2 cells and thus, the currently observed intensity signals are not verifiable. The procedure is currently being optimized for temperature and initial wash buffer conditions, with 10% formamide and 3X dextran sulfate being used.
Caco-2 cells are currently being optimized for successful imaging of exons 2, 4, 5, and 6. Future work and testing of various wash buffer conditions will determine the optimal conditions for imaging.