Multiplexing clonality: combining RGB marking and genetic barcoding

Abstract

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

“…Two independent duplex PCRs were designed that detect sequences within either the 5 0 SIN-LTR or the internal SFFV promoter of the LeGO vector in conjunction with a reference (REF) gene (murine erythropoietin receptor). 5,44 Vector-specific PCR amplicons were detected using a fluorescein amidite (FAM)-labeled black hole quencher (BHQ) probe, whereas a 6-hexachlorofluorescein (HEX)-labeled BHQ probe was used for the REF gene. Primer and probe sequences are available on request.…”

“…Recent efforts have applied a variety of techniques, including genetic DNA barcoding, 3 transposon-based mutagenesis, 4 fluorescentlabeling approaches, or combinations thereof. [5][6][7] Although genetic DNA barcoding allows labeling of highly complex subpopulations, it is largely restricted to the analysis of lysed cells and requires multiple time-consuming and costly steps, including next-generation sequencing and biostatistical analysis, before the different clonal populations can be quantified. 6 Cell marking with fluorescent proteins, on the other hand, enables tracking of live cells and their quick analysis in and ex vivo.…”

“…5 Wu et al 12 recently proposed an elegant approach to quantify fluorescently labeled clonal compositions by using spectral analysis software that identifies RGB-marked cells in pictures (e.g., tissue sections) based on their color. Fluorescent cell barcoding (FCB) represents another, flow cytometry-based labeling technique, which utilizes fluorescent dyes for staining of fixed cells resulting in a unique signature due to defined fluorescence intensities and emission wavelengths for different samples.…”

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

“…The GFP-BC32 lentiviral vector encoding for a barcode library described previously in Thielecke et al 40 was used for H/F-LV production. These H/F-LVs were used to transduce unstimulated human CD34…”

A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking

“…This design would allow for flow cytometric tracking of color-coded populations as well as next generation sequencing-based assessment of clonality in multiplexed samples, which may be especially powerful when working with HSCs and leukemic stem cells. [2][3][4]33 To this end, we established color coding conditions for murine HSCs and human CB derived CD34 + HSPC and evaluated the in vivo color code distribution in different BM subpopulations at the end of the observation period (Figures 5 and 6). To neglect cell type-specific differences in CSF promoter activity as well as potential variations in expression intensity due to the influence of the activation status of the (e.g., T) cells, gates were first set based on surface marker expression before analyzing color-coded populations therein.…”