IR/MAR Gene Amplification Reagent
Cat. No. IRMAR
Store at -20°C
Material 7.5kbp DNA fragment containing IR and MAR
Quantity 10 µg DNA/vial, 20 µL TE (sterilized)
Storage - 20 degree C Handle with care to avoid DNA degradation by nuclease contamination
Protein expression system in mammalian cells has the great advantage to produce mammalian protein representing proper three-dimensional conformation as well as modifications after translation. The structure found in mature protein is essential in the process of proteomics study and protein engineering. However, levels of protein expression in mammalian cells are relatively low in comparison with the other expression systems using bacteria or insect cells, so that the lower expression levels make difficulties in further application. In order to obtain a high-producer mammalian cell line, multiple copies of an expression vector might be introduced into a host cell.
IR/MAR gene amplification is a technology based on a novel mechanism of gene amplification discovered in cancer cell studies. A DNA fragment containing IR (Initiation Region) and MAR (Matrix Attachment Region) effectively amplifies the copy number of a co-transfected expression vector, leading to high protein production rate. Stable cell lines with high protein expression can be easily obtained by standard transfection methods and drug selection.
- Expression vector preparation
- standard procedure. Linearize expression vector encoding a protein by a proper restriction enzyme; subsequently, purify the vector in
detail of transfection protocol should be optimized by users. A transfection without the “IR/MAR GeneAmplification Reagent” (1.0-2.0 μg) and a linearized expression vector (1.0-2.0 μg). For the best result, the standard transfection reagent, cells cultured up to 70-80% confluency are co-transfected with the “IR/MAR Gene On one day before transfection, plate cells in a well of 6 wells plate. According to the protocol attached to a Amplification Reagent” can work as a negative control to estimate the effect of IR/MAR gene amplification.
- Drug selection
- by drug selection for one month.initial dosage so as to stimulate gene amplification. Stable cell lines with high protein expression can be obtained blasticidin S increases up to 5-20 folds from advance. After double drug selection for a few days, dosage of the selection marker of expression vector and blasticidin S. Effective dosages of drugs should be determined in On the next day of transfection, transfer the cells to 10cm dish; subsequently, start double drug selection using
- 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.
The use of this product is strictly limited for research purpose only in the organization that purchaser is belonged.
The purchaser should not make any copies or derivatives of this product. The purchaser should not transfer and/or resell copies and/or derivatives of this product to any third parties.
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An example of experimental procedure
- Establishment of stable transfectant
- Sub-confluent HEK293 cells were transfected with the “IR/MAR Gene Amplification Reagent” (1.0-2.0 μg) and a inearized expression vector (1.0-2.0 μg). On the next day of the transfection, the transfected cells were transferred to 10cm dish and cultured in double drug selection medium (DMEM+10% FCS, 0.5 mg/mL Neomycin, 10 μg/mL Blasticidin S).The cells were transferred every 4-7 days. Blasticidin S was added at 100 μg/ml (final concentration) in selection medium from the second passage.
- Screening of high producer by RealTime-PCR
- Twelve independent clones derived from CHO cells were isolated as stable transfectants. The relative ratio of mRNA expression of each clone is determined by RealTime-PCR. Mouse β-Actin gene is used as internal standard. In the calculation based on ΔΔCt, the expression level of the highest producer line was 235-fold higher than that of lowest producer.
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