To visualize various processes in a cell, fluorescent protein genes are often introduced into the cell genome. At the same time, the issue of preserving the properties of the initial cell culture, whose phenotype and genotype become significantly different from that of the primary culture, becomes relevant and important. The obtaining of subclones further exposes the phenotype and genotype to additional evolution, especially when working with tumor cells.

THE AIM OF THE STUDY

To analyze the expression level of the fluorescent TagRFP protein in Hep2-TagRFP tumor cells, compare the morphology of clones isolated from a heterogeneous Hep2-TagRFP culture, and identify phenotypic differences compared with the original heterogeneous cell culture.

MATERIALS AND METHODS

The Hep2-TaqRFP laryngeal adenocarcinoma line was obtained using lentiviral transduction. Subsequent cloning and selection allowed the selection of two subclones 2B10 and 2F11 stably expressing TagRFP fluorescent protein. MTT test, cytofluorometry with an analysis of the cell cycle, real-time PCR, tumor xenotransplants developing in nude mice were used to confirm the phenotypic differences in subclones from the original cell line.

RESULTS AND DISCUSSION

A heterogeneous fluorescent cell line of laryngeal adenocarcinoma Hep2 with constitutive expression of the TagRFP protein was obtained. Based on the Hep2-TagRFP line, two stable subclones were obtained and their properties were studied. It has been shown that the high copyicity of the TagRFP transgene can affect the expression of key cytoskeletal genes (ACTB and TUBB3), reduce the level of proliferative activity and change the level of metabolic activity (activity of mitochondrial oxidoreductases), also demonstrating a decrease in tumorigenicity of the obtained clones in nude mice.

CONCLUSION

Random embeddings of the lentiviral cassette can affect the expression of key genes (“household” genes), which has been demonstrated on the obtained clones of tumor cells with a high level of expression of the target protein. The obtained subclones of tumor transductant cells differ from the original heterogeneous culture not only in the level of fluorescence of the TagRFP protein in cells, but also in metabolic, proliferative and tumorogenic properties.