2.2 Transient transfection of the cells

The full-length open reading frame (ORF) of the human DCK gene was subcloned using standard cloning techniques from human cDNA. Cloning oligonucleotide PCR primers were designed to be homologic to the human DCK gene (22 bp part of the gene containing start and stop codon) and also to contain specific sites for restriction enzymes (XhoI and NotI). The resulting PCR product with the ORF of the human DCK gene was then subcloned into the pCIneo expression vector (Promega Corporation, Madison, WI) named as pCIneoDCK. Resulting plasmid was and verified by sequencing to exclude clones with mutations. Human cDNA used as a template for this PCR was prepared in our laboratory from human prostatic tumor cell line PC3. A green fluorescent protein (GFP) expression vector pCIneoGFP was used to determine transfection efficiency as a mock plasmid (kindly provided by G. Margison and J. Libby, Paterson Institute for Cancer Research, Christie Hospital, Manchester). Twenty-four hours prior to transfection with pCIneoDCK and pCIneoGFP vectors, JIMT-1 and T-47D cells were seeded in 24-well plates at a density of 1.5 × 10⁵ cells/well and 2 × 10⁵ cells/well, respectively. All transfections were carried out using 4 μl of FuGENE® HD Transfection Reagent (Promega Corporation) per 1 μg of plasmid DNA according to the manufacturer’s recommendations. After 24 h, the transfection medium was replaced with the fresh cultivation medium containing the selection reagent G418. Transfected cells were selected for 2 weeks with 1 mg/ml G418 (Sigma-Aldrich, Germany) for JIMT-1 and 0.5 mg/ml G418 for T-47D based on the previous G418 concentration testing. Schematic maps of the vectors pCIneoDCK and pCIneoGFP are presented in Supplementary Figure S1.