RGB marking with lentiviral vectors for multicolor clonal cell tracking

Weber, Kristoffer; Thomaschewski, Michael; Benten, Daniel; Fehse, Boris

doi:10.1038/nprot.2012.026

Cited by 82 publications

(112 citation statements)

References 23 publications

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“…In fact, the deceleration of the cells in the course of migration ( Figures 4A and 7E) and existence of a number of additional criteria (e.g., the soma size, the direction of the leading process as indicator of migration direction, the ratio of the fluorescence recorded in different channels (proportional to the number of incorporated virus copies and the strength of FP expression) and the existence of stationary cells in the neighborhood) reduce the ambiguity in the system even further. It has to be noticed that the RGB-marking approach is not restricted to seven colors and, as shown previously [21,26], can provide a large number of different color hues. The precise readout of these hues, however, requires more sophisticated imaging and color-identification approaches, which seem unnecessary within the framework of the present study.…”

Section: Discussionmentioning

confidence: 99%

“…The relative fraction of cells labeled with a given color depends on many factors including viral titer, diffusion properties after injection, cell cycle status, expression of anti-viral restriction factors, etc. [26,27]. Data collected from 6 mice (34-136 cells per mouse) indicated that under our experimental conditions, around a quarter of FP-positive JGNs were triple-labeled (23.7 ± 3.6%, mean ± SEM), 6-19% double-labeled (GB 18.9 ± 15.2%; RB 6.5 ± 11.3%; RG 12.2 ± 6.6%) and 7-16% were labeled by a single color (G 9.0 ± 7.5%; B 16.2 ± 15.3%; R 7.2 ± 9.9%; Figure 2C).…”

Section: Ocps Reveals An Active Migration Of Adult-born Neurons In Thmentioning

confidence: 99%

“…In some sets of experiments, retrovector encoding eGFP driven by CAG [44] was utilized and packaged by 1F8 cells derived from 293GPG cell line [45]. Supernatants containing retroviral particles were concentrated about 100-fold by centrifugation at 8 000× g and 6 °C for 8-12 h. Concentrated supernatants were titrated [26] on 293T cells. For titration, 293T cells were incubated at 5 × 10 4 cells in 0.5-ml medium in each well of a 24-well plate in the presence of 8 µg/ml polybrene.…”

Section: Retroviral Vector Productionmentioning

confidence: 99%

“…de. Cell-free supernatants containing viral particles were produced by transient transfection of HEK-293T-packaging cells as described previously [26,43]. Retroviral vectors have been packaged using pcDNA3.MLVgp and phCMV-VSV-G.…”

Section: Retroviral Vector Productionmentioning

confidence: 99%

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Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb

Liang

Riecken

et al. 2016

Cell Res

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The behavior of adult-born cells can be easily monitored in cell culture or in lower model organisms, but longitudinal observation of individual mammalian adult-born cells in their native microenvironment still proves to be a challenge. Here we have established an approach named optical cell positioning system for long-term in vivo single-cell tracking, which integrates red-green-blue cell labeling with repeated angiography. By combining this approach with in vivo two-photon imaging technique, we characterized the in vivo migration patterns of adult-born neurons in the olfactory bulb. In contrast to the traditional view of mere radial migration of adult-born cells within the bulb, we found that juxtaglomerular cells switch from radial migration to long distance lateral migration upon arrival in their destination layer. This unique long-distance lateral migration has characteristic temporal (stop-and-go) and spatial (migratory, unidirectional or multidirectional) patterns, with a clear cell age-dependent decrease in the migration speed. The active migration of adult-born cells coincides with the time period of initial fate determination and is likely to impact on the integration sites of adult-born cells, their odor responsiveness, as well as their survival rate.

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Section: Discussionmentioning

confidence: 99%

Section: Ocps Reveals An Active Migration Of Adult-born Neurons In Thmentioning

confidence: 99%

Section: Retroviral Vector Productionmentioning

confidence: 99%

Section: Retroviral Vector Productionmentioning

confidence: 99%

See 2 more Smart Citations

Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb

Liang

Riecken

et al. 2016

Cell Res

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“…8,9 The number of exploitable fluorescent colors can be significantly increased by applying combinatorial labeling strategies such as red-green-blue (RGB) marking. 10,11 The latter is based on the transduction of target cells with integrating retro-/lentiviral vectors that mediate highly variable expression of random combinations of these three basic colors (RGB), thus generating all possible color combinations. However, RGB marking does not permit precise quantification or recovery of single clones out of complex and heterogeneous subpopulations by fluorescent-activated cell sorting (FACS).…”

Section: Introductionmentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

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Intratumoral heterogeneity has been identified as one of the strongest drivers of treatment resistance and tumor recurrence. Therefore, investigating the complex clonal architecture of tumors over time has become a major challenge in cancer research. We developed a new fluorescent "optical barcoding" technique that allows fast tracking, identification, and quantification of live cell clones in vitro and in vivo using flow cytometry (FC). We optically barcoded two cell lines derived from malignant glioma, an exemplary heterogeneous brain tumor. In agreement with mathematical combinatorics, we demonstrate that up to 41 clones can unambiguously be marked using six fluorescent proteins and a maximum of three colors per clone. We show that optical barcoding facilitates sensitive, precise, rapid, and inexpensive analysis of clonal composition kinetics of heterogeneous cell populations by FC. We further assessed the quantitative contribution of multiple clones to glioblastoma growth in vivo and we highlight the potential to recover individual viable cell clones by fluorescence-activated cell sorting. In summary, we demonstrate that optical barcoding is a powerful technique for clonal cell tracking in vitro and in vivo, rendering this approach a potent tool for studying the heterogeneity of complex tissues, in particular, cancer.

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Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Möller

Bhat

Riecken

et al. 2019

Biotech & Bioengineering

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The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through the cell cycle causes metabolic regulations with variable productivities in antibody‐producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle‐derived population dynamics. In this study, process‐induced cell cycle synchronization was assessed in repeated‐batch and fed‐batch cultures. An automated flow cytometry set‐up was developed to measure the cell cycle distribution online, using antibody‐producing CHO DP‐12 cells transduced with the cell cycle‐specific fluorescent ubiquitination‐based cell cycle indicator (FUCCI) system. On the basis of the population‐resolved model, feeding‐induced partial self‐synchronization was predicted and the results were evaluated experimentally. In the repeated‐batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed‐batch process with up to 25×106 cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high‐frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle‐dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.

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RGB marking with lentiviral vectors for multicolor clonal cell tracking

Cited by 82 publications

References 23 publications

Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb

Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

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