Meeting Abstract

P1-39   -   Development of a fluorescent nuclear marker for quantification of live tilapia cells over time. Le, TK*; Hamar, JC; Kültz, D; University of California, Davis; University of California, Davis; University of California, Davis trkle@ucdavis.edu

Cell cultures are proficient models for mechanistic studies of which our lab utilizes a tilapia brain cell line (OmB). A desired phenotype to measure is proliferation quantified by periodic automated counting of a cell population. Our current microscope can quantify nuclei with a nuclear stain (DAPI) because of well-defined boundaries despite high confluency and overlapping cells but at the cost of cell mortality after treatment. Enhanced green fluorescent protein (EGFP) would be an alternative to avert the mortality issue, but unmodified EGFP distributes throughout the cell making distinction between adjacent cells difficult. Past work in our lab attempted to concentrate EGFP in the nucleus by adding a SV40 nuclear localization signal (NLS) to EGFP. The new fusion protein (EGFP-NLS) showed enriched nuclear EGFP but retained significant EGFP in the cytoplasm. We hypothesized addition a degradation signal to EGFP-NLS would reduce cytoplasmic accumulation of EGFP making the nucleus more defined. In other reports, addition of the human cyclin B1 destruction box (DB) sequence to fluorescent proteins limited accumulation. An alignment between the human Cyclin B1 DB amino acid sequence (AAs) with a tilapia Cyclin B1 AAs determined the tilapia DB was located in exon 2 of the gene. A new fusion protein (CDB-EFGP-NLS) coding sequence was made by fusing the tilapia Cyclin B1 exon 2 nucleotide sequence to the EGFP-NLS sequence and then cloned into an expression vector. Cytoplasmic EGFP fluorescence was less in OmB cells transfected with the CDB-EFGP-NLS vector compared to the EFGP-NLS vector increasing distinction between nuclei of different cells. Integration of CDB-EGFP-NLS into the genome of OmB cells will produce a new cell line that can be used for proliferation assays in which the same live cells can be quantified over time. Funded by NSF-IOS 1656371 and BARD IS-5358-21.