Expression data from transfected HEK cells

Many missense variants with associated phenotypes lie in regions of proteins that are not well understood. However, often a putative function can be derived through study of related sequences that have known functional information in the respective protein region. We used a simple transfection & microarray gene expression analysis to study such a missense variant, finding that differentially regulated genes hint at an increased activity of the protein of interest. Cells were seeded into wells of a 6-well plate (Day 1). The next day cells were transfected with a pDEST30 vector (2.5ng) containing the coding sequence of the protein of interest (MAP2K3) as WT or mutant (A84T) (Day2). One day later we added selection pressure (350 µg/ml Geneticin) to the mixture (Day 3). Cells were selected and compared to the neg. control. Typically cells would be induced on Day 7 for 24 h with 1 µg/ml Tetracycline and harvested one day later for total RNA extraction. cDNA libraries were prepared and analyzed by the DKFZ Genomics core facility, according to the standard operating protocols.