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

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 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.

SUBMITTER: Mosaad EO

PROVIDER: S-EPMC6683182 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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

Mosaad E O EO Futrega K K Seim I I Gloss B B Chambers K F KF Clements J A JA Doran M R MR

Scientific reports 20190805 1

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 a ...[more]

PMID: 31383876

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Project description:BackgroundPlacental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts.Methodology/principal findingsZona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2)/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages.Conclusions/significanceThese results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero.

Project description:BACKGROUND:In the early phase after a total knee arthroplasty (TKA), patients experience multi-level weakness in the operated leg, which is caused primarily by reduced central nervous system (CNS) activation failure of the muscles - especially the knee extensors (quadriceps muscle). Whether similar levels of neuromuscular activity of the muscles in the operated leg, elicited during strength training exercises in machines, can be reached during strength training exercises in more simple forms is unknown. Many clinicians are faced with the problem of not having strength training equipment at their institution or having to prescribe simple strength training exercises for home-based training. Therefore, the purpose of this study was to determine which strength training exercises that activated the muscles in the operated leg the most after TKA. The hypothesis was that strength training exercises performed in machines would elicit higher levels of voluntary peak quadriceps and hamstring muscle activity than strength training exercises performed in more simple forms, using elastic bands or the patients' own body weight. METHODS:A cross-sectional electromyographic study investigated voluntary peak muscle activity in the operated leg during 6 different strength training exercises. Twenty-four patients, who received a TKA 4 to 8?weeks earlier, performed the exercises in a randomized order, using a pre-determined loading of 10 RM (repetition maximum). Voluntary peak muscle activity (%EMGmax) was calculated for the quadriceps and hamstring muscles for each exercise. RESULTS:Knee extensions with elastic band showed significantly higher voluntary peak quadriceps muscle activity than knee extensions in machine (93.3 vs. 74.9; mean difference, 18.3 %EMGmax [95% confidence interval (CI), 11.7 to 24.9]; P?<?0.0001). Similarly, one-legged squat (and sit to stand) elicited higher voluntary peak quadriceps muscle activity than leg press in machine (86.7 vs. 66.8; mean difference, 19.9 %EMGmax [95% CI, 14.8 to 25.0]; P?<?0.0001). CONCLUSIONS:Strength training exercises in more simple forms elicited higher voluntary peak quadriceps muscle activity than strength training exercises in machines early after TKA. Consequently, simple home-based strength training exercises using e.g. elastic bands or the patients' own bodyweight should be considered to alleviate muscle strength losses early after TKA. TRIAL REGISTRATION:ClinicalTrials.gov identifier: NCT01708980 .

Dataset Information

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

Publications

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

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