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.

Figure 1.

Vector construction and deciphering the color-code by single-cell PCR. (a) Barcodes with color signatures were introduced in front of the 3′ LTR of the respective LeGO vector by recombinant DNA technology. The barcodes consist of 16 random nucleotides separated by fixed triplets, the pattern of triplets cipher for the respective fluorescent protein in the vector. In all, 10¹⁰ plasmids of each plasmid library were used for NGS via Hiseq 2000 (Illumina). For all three RGB vectors, even distribution of the 4 nt reflecting comprehensive randomization during synthesis was found (based on at least 25 million reads for each vector). (b) For RGB transduction of HEK293T cells, equal amounts of viral particles of the LeGO-C2-BC16 (red), LeGO-V2-BC16 (yellow-green) and LeGO-Cer2-BC16 (cyan-blue) were used. (c–e) Two identically colored single cells from a pink colony were consecutively picked with the CellCelector. (f) Single-cell PCR generates a barcode-containing fragment of 229 bp for both cells picked in (d) and (e). (g) After subcloning of the PCR fragment into plasmids, sequencing of individual clones revealed a barcode signature of one blue and two red viral copies in each of the picked cells, which is in good agreement with the pink color of the analyzed clone following the additive color model.

Figure 2.

Analyzing single cells from regenerated liver tissue. (a) LeGO-C2-BC16 (red), LeGO-V2-BC16 (yellow-green) and LeGO-Cer2-BC16 (cyan-blue) were used for transduction of primary murine hepatocytes. Transduced cells were transplanted intrasplenically into hemizygous uPA/SCID mice. Liver sections taken 4 weeks post-transplantation show a regeneration of the liver with RGB-marked patches. (b) Schematic representation of the experimental procedure: laser dissection of single cells from cryosections of the regenerated liver, single-cell PCR, subcloning of the obtained PCR fragment and sequencing of the obtained clones. (c, d) Laser dissection of one single cell from the liver section. (e) Sequencing of bacterial clones revealed a single barcode with the LeGO-Cer2-BC16 signature encoding for the Cerulean fluorescence protein.

Figure 3.

Barcodes for tracking leukemia. (a) Schematic representation of vector ΔTrkA-LeGO-iG2-BC16 co-expressing the oncogene ΔTrkA and eGFP and equipped with a GFP-BC16 barcode library. The barcode library consisting of >700 000 different plasmids showed an equal distribution of the randomized nucleotides as evident from Illumina sequencing (>26 Mio reads) on 10¹⁰ plasmids (illustrated in the frequency plot). (b) Viral supernatant of the ΔTrkA-LeGO-iG2-BC16 plasmid library was used to transduce syngeneic lineage-negative bone marrow cells from male donors. Transduced cells were transplanted into lethally irradiated female recipient mice (n = 4). Control mice (n = 4) were transplanted with a barcoded eGFP marking vector (LeGO-G2-BC16). During follow-up, blood was taken every 4 weeks from transplanted mice. One mouse developed full-blown leukemia after 19 weeks as evidenced by the high proportion of eGFP-positive cells in the blood. All other mice showed stable eGFP counts in the peripheral blood during follow-up analysis. (c) Frequency analysis (stacked box plot) for barcodes found in leukemia samples by NGS of DNA from blood, spleen and bone marrow cells. The 10 most abundant barcodes were given individual colors, all other barcode sequences are summarized by gray boxes. (d) Sequences of the three leukemia-contributing barcodes, wobble bases are marked in accordance with the color of the respective box plot in (c).