Constraints to counting bioluminescence producing cells by a commonly used transgene promoter and its implications for experimental design
| Autor: | Karen F. Chambers, Michael R. Doran, Brian S. Gloss, Katarzyna Futrega, Judith A. Clements, Inge Seim, Eman Mohamed Othman Mosaad |
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| Rok vydání: | 2019 |
| Předmět: |
Male
Cancer microenvironment 0301 basic medicine Stromal cell Population Cell lcsh:Medicine Cellular imaging Biology Article 03 medical and health sciences 0302 clinical medicine Genes Reporter Cell Line Tumor medicine Humans Bioluminescence Luciferase Transgenes Luciferases Promoter Regions Genetic Cancer models lcsh:Science education Reporter gene education.field_of_study Multidisciplinary lcsh:R Prostatic Neoplasms Mesenchymal Stem Cells Coculture Techniques Cell biology Gene Expression Regulation Neoplastic Luminescent Proteins 030104 developmental biology medicine.anatomical_structure Cell culture Luminescent Measurements Cancer cell lcsh:Q Gene expression 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-12 (2019) Scientific Reports |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-019-46916-z |
| Popis: | 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 the low luciferase gene copy number and genome insertion locations likely contributed to the reporter gene expression being exquisitely sensitive BMSC paracrine signals. In this study, we show that it is possible to generate a range of stable and reliable luciferase reporter prostate- and breast- cancer cell populations but advise not to assume stability across different culture conditions. Reporter stability should be validated, on a case-by-case basis, for each cell line and culture condition. |
| Databáze: | OpenAIRE |
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