Constraints to counting bioluminescence producing cells by a commonly used transgene promoter and its implications for experimental design

Scientific Reports
E O MosaadM R Doran

Abstract

It is routine to genetically modify cells to express fluorescent or bioluminescent reporter proteins to enable tracking or quantification of cells in vitro and in vivo. Herein, we characterized the stability of luciferase reporter systems in C4-2B prostate cancer cells in mono-culture and in co-culture with bone marrow-derived mesenchymal stem/stromal cells (BMSC). An assumption made when employing the luciferase reporter is that the luciferase expressing cell number and bioluminescence signal are linearly proportional. We observed instances where luciferase expression was significantly upregulated in C4-2B cell populations when co-cultured with BMSC, resulting in a significant disconnect between bioluminescence signal and cell number. We subsequently characterized luciferase reporter stability in a second C4-2B reporter cell line, and six other cancer cell lines. All but the single C4-2B reporter cell population had stable luciferase reporter expression in mono-culture and BMSC co-culture. Whole-genome sequencing revealed that relative number of luciferase gene insertions per genome in the unstable C4-2B reporter cell population was lesser than stable C4-2B, PC3 and MD-MBA-231 luciferase reporter cell lines. We reasoned that t...Continue Reading

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Citations

Feb 27, 2020·Pharmaceutics·Nina BonoGabriele Candiani

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Datasets Mentioned

BETA
M15077.1

Methods Mentioned

BETA
flow cytometry
PCR

Software Mentioned

Prism
BWA
FlowJo
Primer3Plus
TreeStar
- BLAST
STAR
MEM
GraphPad
Primer

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