Research Reagent Only

IR/MAR Gene Amplification Reagent

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, levelsof 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 (MatrixAttachment Region) effectively amplifies the copy number of a co-transfected expression vector, leading to highprotein production rate. Stable cell lines with high protein expression can be easily obtained by standardtransfection methods and drug selection.

Material
Quantity
Storage

7.5kbp DNA fragment containing IR and MAR
10 µg DNA/vial, 20 µL TE (sterilized)- 20 degree C
Handle with care to avoid DNA degradation by nucleasecontamination

Structure of reagent

Promoter

BSRBSRIRMARIRMAR: Blasticidin S deaminase from Escherichia coli
: Mammalian replication initiation region: Matrix Attachment Region【Protocol】
1. Expression vector preparation
Linearize expression vector encoding a protein by a proper restriction enzyme; subsequently, purify the vector instandard procedure.

2. Transfection
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, thedetail 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 inadvance. After double drug selection for a few days, dosage of blasticidin S increases up to 5-20 folds frominitial dosage so as to stimulate gene amplification. Stable cell lines with high protein expression can be obtainedby 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 numberof an expression vector as well as mRNA expression levels is the possible indicator, which is determined byrealtime-PCR analysis.
【References】
N.Shimizu et al. Cancer Res 61, 6987–6990, 2001
N.Shimizu et al. Cancer Res 63, 5281–5290, 2003
N.Shimizu et al. Exp Cell Res 302(2), 233-2433, 2005
N.Shimizu et al. Nucleic Acids Res 33(19), 6296-6307, 2005
T.Hashidume et al. J Cell Biochem 101, 552-565, 2007
【License statements】
This product and IR/MAR gene amplification technology are covered by the claims of JP Patent No. 3755028,3882042 and 2011-019563.
The use of this product is strictly limited for research purpose only in the organization that purchaser isbelonged.
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.
In the case of order, please check the License statements and submit the signed License Agreement toTransGenic Inc.
Manufacturer

7-1-14Minatojimaminami-machi, Chuo-ku, Kobe, Japan 650-0047
Telephone: +81-78-306-0295
URL:http://www.transgenic.co.jp

FAX:+81-78-306-0296
techstaff@transgenic.co.jp

ORF

IR/MAR

Expression
vector

IR/MARsequence【An example of experimental procedure】
1. Establishment of stable transfectant
Sub-confluent HEK293 cells were transfected with the “IR/MAR
Gene Amplification Reagent” (1.0-2.0 μg) and a linearized expression

Co-Transfection

vector (1.0-2.0 μg).
On the next day of the transfection, the transfected cells were
Host cell linetransferred to 10cm dish and cultured in double drug selection medium(DMEM+10% FCS, 0.5 mg/mL Neomycin, 10 μg/mL Blasticidin S). TheDrug selection(1-2 months)

cells were transferred every 4-7 days. Blasticidin S was added at 100 μg/ml(final concentration) in selection medium from the second passage.

Stable transfectant
Cloning/ScreeningHigh producer

2. 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 asinternal standard. In the calculation based on ΔΔCt, the expression level of the highest producer line was235-fold higher than that of lowest producer.
8Clone.1

6

Clone.2
Clone.3
Clone.4
Clone.5

High producer

Clone.6

Intensity

4

Host cell

Clone.7
Clone.8
Clone.92

Low producer

Clone.10
Clone.11
Clone.12
Mock0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40-2
CyclesAmplification curve of integrated gene
The relative ratio of mRNA expression is determined by RealTime-PCRMock