1. Expression vector preparation
Linearize expression vector encoding a protein by a proper restriction enzyme; subsequently, purify the vector in standard procedure.
On one day before transfection, plate cells in a well of 6 wells plate. According to the protocol attached to a standard transfection reagent, cells cultured up to 70-80% confluency are co-transfected with the “IR/MAR Gene Amplification Reagent” (1.0-2.0 μg) and a linearized expression vector (1.0-2.0 μg). For the best result, the detail of transfection protocol should be optimized by users. A transfection without the “IR/MAR Gene Amplification Reagent” can work as a negative control to estimate the effect of IR/MAR gene amplification.
3. Drug selection
On the next day of transfection, transfer the cells to 10cm dish; subsequently, start double drug selection using the selection marker of expression vector and blasticidin S. Effective dosages of drugs should be determined in advance. After double drug selection for a few days, dosage of blasticidin S increases up to 5-20 folds from initial dosage so as to stimulate gene amplification. Stable cell lines with high protein expression can be obtained by drug selection for one month.
4. Cloning of high producer
Limiting dilution method can be used to obtain high producer. As a procedure to select clones, the copy number of an expression vector as well as mRNA expression levels is the possible indicator, which is determined by realtime-PCR analysis.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.