Multiplexing clonality: combining RGB marking and genetic barcoding

Kerstin Cornils,Lars Thielecke,Ingo Roeder,Boris Fehse,Nadja Kleist,Ingmar Glauche,Tassilo Volz,Michael Forgber,Kais Hussein,Michael Thomaschewski,Sebastian Gerdes,Kristoffer Riecken,Andreas Dahl,Svenja Hüser,Maura Dandri

doi:10.1093/nar/gku081

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.

Highlights

Permanent cell marking by integrating vectors has been used to track cell populations or even single cells in vitro and in vivo [1]
Multi-color marking techniques have been introduced based on complex recombination strategies (‘Brainbow’ imaging) [6] or simultaneous transduction with different lentiviral vectors (‘RGB marking’) [7] that allow for the phenotype-based identification of differently marked cells down to the clonal level
In proof-of-principle experiments, we show that fluorescent cell marking with barcoded LeGO vectors facilitates clonal analysis both in vitro and in vivo, in models of normal tissue regeneration as well as malignant outgrowth

Summary

Introduction

Permanent cell marking by integrating (retroviral) vectors has been used to track cell populations or even single cells in vitro and in vivo [1]. Cell marking studies have provided important insights into biology and development of cells, tissues, organs and even whole organisms [2]. For many years, gene marking has been considered one of the most successful approaches in human gene therapy [3]. The cloning and successful expression of Aequorea victoria green fluorescent protein (GFP), first described in the 1970s, facilitated direct visualization of genemarked cells and initiated a new boost of marking approaches in experimental biology and biomedicine [2,4]. Based on the subsequent cloning of further fluorescent proteins, interactions of differently labeled cell populations could be studied [5]. Multi-color marking techniques have been introduced based on complex recombination strategies (‘Brainbow’ imaging) [6] or simultaneous transduction with different lentiviral vectors (‘RGB marking’) [7] that allow for the phenotype-based identification of differently marked cells down to the clonal level

Methods

Results

Conclusion