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<p>To visualize different processes in the cell, the genes of fluorescent proteins are often introduced into the cell genome. At the same time, the issue of stability of the initial cell culture, the phenotype-genotypic properties of which undergo significant transformations, is relevant. The selection of subclones is even more associated with artificial oncotransformation, which highlights the importance in the context of cancer studies. <b>Aim of the study</b>. To analyze the level of expression of fluorescent TagRFP protein in Hep2-TagRFP tumor cells, compare morphology of the clones isolated from heterogeneous Hep2-TagRFP culture, and identify phenotypic differences as compared with the initial heterogeneous cell culture. <b>Material and Methods.</b> The Hep2-TaqRFP laryngeal adenocarcinoma line was obtained using lentiviral transduction. Subsequent cloning and selection allowed us to select two subclones 2B10 and 2F11 that stably express the fluorescent TagRFP protein. MTT test, cytofluorometry with the analysis of cell cycle, real-time PCR, obtaining xenografts on Nude line mice were used to confirm the phenotypic differences of the subclones from the initial cell line. <b>Results and Discussion.</b> A heterogeneous fluorescent Hep2 laryngeal adenocarcinoma cell line 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 was demonstrated that a high copy number of the TagRFP transgene can affect the expression of key cytoskeleton genes (<i>ACTB</i> and <i>TUBB3</i>), decrease the level of proliferative activity, and change the level of metabolic one (activity of mitochondrial oxidoreductases), also demonstrating a decrease in tumorigenicity of the obtained clones in Nude line mice. <b>Conclusions.</b> Random insertions of lentiviral cassette can affect the expression of key genes (“housekeeping” genes), which was demonstrated on the obtained tumor cell clones with a high level of target protein expression. The obtained subclones of tumor transductant cells differ from the initial heterogeneous culture not only by the level of fluorescence of the TagRFP protein in the cells, but also by metabolic, proliferative, and tumorigenic properties.</p>

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Reduction in the Expression of Cytoskeletal Genes in Tumor Cells during Lentiviral Transduction

  • 期刊论文

  • 发表日期：

    2025年3月1日

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  • V. V. Zherdeva,
  • N. V. Rassomakhina,
  • A. Yu. Yurkova,
  • M. V. Monakhova,
  • E. A. Guck,
  • U. A. Apukhtina,
  • L. G. Maloshenok

摘要

To visualize different processes in the cell, the genes of fluorescent proteins are often introduced into the cell genome. At the same time, the issue of stability of the initial cell culture, the phenotype-genotypic properties of which undergo significant transformations, is relevant. The selection of subclones is even more associated with artificial oncotransformation, which highlights the importance in the context of cancer studies. Aim of the study. To analyze the level of expression of fluorescent TagRFP protein in Hep2-TagRFP tumor cells, compare morphology of the clones isolated from heterogeneous Hep2-TagRFP culture, and identify phenotypic differences as compared with the initial heterogeneous cell culture. Material and Methods. The Hep2-TaqRFP laryngeal adenocarcinoma line was obtained using lentiviral transduction. Subsequent cloning and selection allowed us to select two subclones 2B10 and 2F11 that stably express the fluorescent TagRFP protein. MTT test, cytofluorometry with the analysis of cell cycle, real-time PCR, obtaining xenografts on Nude line mice were used to confirm the phenotypic differences of the subclones from the initial cell line. Results and Discussion. A heterogeneous fluorescent Hep2 laryngeal adenocarcinoma cell line 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 was demonstrated that a high copy number of the TagRFP transgene can affect the expression of key cytoskeleton genes (ACTB and TUBB3), decrease the level of proliferative activity, and change the level of metabolic one (activity of mitochondrial oxidoreductases), also demonstrating a decrease in tumorigenicity of the obtained clones in Nude line mice. Conclusions. Random insertions of lentiviral cassette can affect the expression of key genes (“housekeeping” genes), which was demonstrated on the obtained tumor cell clones with a high level of target protein expression. The obtained subclones of tumor transductant cells differ from the initial heterogeneous culture not only by the level of fluorescence of the TagRFP protein in the cells, but also by metabolic, proliferative, and tumorigenic properties.