We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.

Cells transduced with lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. We have recently extended this principle of clonal cell marking by introducing the method of RGB marking, which makes use of the simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. In accordance with the additive color model, individual RGB-marked cells display a large variety of unique and highly specific colors. Color codes remain stable after cell division and can thus be used for clonal tracking in vivo and in vitro. Our protocol for efficient RGB marking is based on established methods of lentiviral vector production (3-4 d) and titration (3 d). The final RGB-marking step requires concurrent transduction with the three RGB vectors at equalized multiplicities of infection (1-12 h). The initial efficiency of RGB marking can be assessed after 2-4 d by flow cytometry and/or fluorescence microscopy.

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.

The LTB curriculum constitutes a new highly standardized and proficiency level-based training program for basic skills in MIS. Transferability of the task content to a (sub)-realistic environment could be demonstrated. Still, future trials will have to further validate the effectiveness of the LTB curriculum.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

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<b><i>Background:</i></b> The use of stereoscopic laparoscopic systems in minimally invasive surgery (MIS) allows a three-dimensional (3D) view of the surgical field, which improves hand-eye coordination. Depending on the stereo base used in the construction of the endoscopes, 3D systems may differ regarding the 3D effect. Our aim was to investigate the influence of different stereo bases on the 3D effect. <b><i>Methods:</i></b> This was a prospective randomized study involving 42 MIS-inexperienced study participants. We evaluated two laparoscopic 3D systems with stereo bases of 2.5 mm (system A) and 3.8 mm (system B) for differences in learning MIS skills using the Lübeck Toolbox (LTB) video box trainer. We evaluated participants’ performance regarding the times and repetitions required to reach each exercise’s goal. After completing the final exercise (“suturing”), participants performed the exercise again using a two-dimensional (2D) representation. Additionally, we retrospectively compared our study results with a preliminary study from participants completing the LTB curriculum with a 2D system. <b><i>Results:</i></b> The median number of repetitions until reaching the goals for LTB exercises 1, 2, 3, and 6 for system A were: 18 (range 7–53), 24 (range 8–46), 24 (range 13–51), and 21 (range 10–46), respectively, and for system B were: 12 (range 2–30), 16 (range 6–43), 17 (range 4–47), and 15 (range 6–29), respectively (<i>p</i> = not significant). Changing from a 3D to a 2D representation after completing the learning curve led to a longer average time required, from 95.22 to 119.3 s (<i>p</i> &#x3c; 0.0001), for the last exercise (exercise 6; “suturing”). When comparing the results retrospectively with the learning curves acquired with the 2D system, there was a significant reduction in the number of repetitions required to reach the LTB exercise goals for exercises 1, 3, and 6 using the 3D system. <b><i>Conclusion:</i></b> Stereo bases of 2.5 and 3.8 mm provide acceptable bases for designing 3D systems. Additionally, our results indicated that MIS basic skills can be learned quicker using a 3D system versus a 2D system, and that when the 3D effect is eliminated, the corresponding compensatory mechanisms must be relearned.

As an addition to the regular undergraduate program, the Lübeck Toolbox Curriculum was well accepted by many students. Evaluation showed exceedingly positive results. Furthermore, the data suggest that the LTB Curriculum may increase the interest in a surgical specialty among medical students. This aspect seems to be relevant in times where surgeons should make every effort to recruit young doctors for surgical residency.