Publikacja

36th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University, February 21-26 2009

Production of cell lines with stable overexpression of reporter genes and heme oxygenase-1

Jakub Kołodziejski¹, Jerzy Kotlinowski¹, Paweł Dominik¹, Milena Dubiel¹, Magdalena Kozakowska¹, Halina Waś¹, Krzysztof Lemke³, Anna Zebzda², Marcin Majka², Józef Dulak¹, Alicja Józkowicz¹

¹ Department of Medical Biotechnology, Faculty of Biochemistry, Biophisics and Biotechnology, Jagiellonian University, Kraków, Poland.
² Institute of Transplantology, Colegium Medicum, Jagiellonian University, Kraków, Poland.
³ Adamed Sp. z o.o., Pieńkow, Poland.

Introduction.

Retroviral vectors (RVs) due to their ability to integrate viral cDNA into host genome are one of  the most popular tools for gene transfer. RVs are widely used in various studies associated with genetic transformation and gene therapy approaches both in vitro and in vivo. The aim of this study was to produce cell lines with stable overexpression of reporter genes and heme oxygenase-1 to evaluate HO-1 role in wound healing and carcinogenesis.

Methods and Results. In our experiments we used two systems of RVs production: Phoenix-Ampho packaging cells for modification of murine myoblast cell line (C2C12) and PT67 packaging cells for gene transfer into murine melanoma cell line (B16F10). RVs were collected from culture medium after cotransfection of packaging cells with backbone and helper plasmids. In order to produce C2C12 and B16F10 cells with overexpression of luciferase, GFP and HO-1 we performed two-step genetic modification: first we used backbone plasmid encoding reporter genes (pBMN-Luc-I-GFP) and later pLNCX2-HO-1. 

Both cell lines were characterized by very strong expression of reporter genes: luciferase activity was 1000 times higher in genetic modified C2C12 and B16F10 than in wild type cells. What is more despite presence of IRES sequence in our construct GFP fluorescence was strong and let us select positive cells by FACS. Higher expression of HO-1 was detected both on mRNA and protein level, in addition to that we also observed upregulation of HO-1 activity. Above all, expression of all transgenes is stable and do not diminish in the course of time, it is present after months of constant culture. Expression of luciferase unable us to monitor tumor formation after intravenous injection of modified B16F10 cells into mice. Based on the chemiluminescent reaction we are able to trace down B16F10 cells not only in lungs were the biggest tumors were observed but also in other tissues.

Conclusion. Retroviral vectors are very potent transgene carriers which are suitable for permanent modification of mammalian cells. Upon infection with RVs we obtained two cell lines with long lasting expression of genes of interest which are very helpful in other experiments.