Dergiler / Turkish Journal of Medical Sciences / 2011 / Cilt: 41 - Sayı: 5
An efficient method for stable transfection of mouse myogenic C2C12 cell line using a nonviral transfection approach
- Sayfa
- 821–825
- DOI
- —
Özet
To describe in detail the use of the T-RExTM System (a tetracycline-regulated mammalian expression system; Invitrogen) for rapid, easy, and effective generation of stable C2C12 cell clones. Tetracycline-inducible stable cell lines are useful tools to study the function of various genes in different types of mammalian cells. However, the generation of stable cell lines is a time-consuming, technically difficult, and expensive process. Materials and methods: Generation of a stable C2C12 cell line was performed by stable transfection of the cells with the pcDNA6/TR plasmid vector, which contains the tetracycline repressor and the blasticidin drug resistance genes. The establishment of the stable cell line and the efficiency of the tetracycline-inducible gene expression system were shown with the transient transfection of reporter plasmid pTO-EGFP and observation of the GFP expression by fluorescence microscopy. Results: We established a stable T-RExTM C2C12 cell line that can be used for muscle research studies by transfecting the gene of interest with a second plasmid. Conclusion: The T-RExTM system is useful in the development of stable cell lines for protein production or regulation of gene expression.
Abstract
To describe in detail the use of the T-RExTM System (a tetracycline-regulated mammalian expression system; Invitrogen) for rapid, easy, and effective generation of stable C2C12 cell clones. Tetracycline-inducible stable cell lines are useful tools to study the function of various genes in different types of mammalian cells. However, the generation of stable cell lines is a time-consuming, technically difficult, and expensive process. Materials and methods: Generation of a stable C2C12 cell line was performed by stable transfection of the cells with the pcDNA6/TR plasmid vector, which contains the tetracycline repressor and the blasticidin drug resistance genes. The establishment of the stable cell line and the efficiency of the tetracycline-inducible gene expression system were shown with the transient transfection of reporter plasmid pTO-EGFP and observation of the GFP expression by fluorescence microscopy. Results: We established a stable T-RExTM C2C12 cell line that can be used for muscle research studies by transfecting the gene of interest with a second plasmid. Conclusion: The T-RExTM system is useful in the development of stable cell lines for protein production or regulation of gene expression.