Hyper Protein Expression for Mammalian Cells

TG-Sure Expression (IR/MAR)


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Background

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.

< Protocol >  Click here

Experimental example

< Protocol and schematic mechanism of TG-Sure Expression (IR/MAR)>
Sub-confluent HEK293 cells were transfected with the “IR/MAR Gene Amplification Reagent” (1.0-2.0 μg) and a linearized 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 Ct, the expression level of the highest producer line was 235-fold higher than that of lowest producer.


Amplification curve of integrated gene
The relative ratio of mRNA expression is determined by RealTime-PCR

 

< FISH : Fluorescence in situ hybridization >

Fluorescent photomicrograph of high expression cell constructed by "IR/MAR Gene Amplification Reagent"
Plasmid sequence was introduced into CHO-DG44 by IR/MAR method and the introduction result was confirmed by FISH.
  Red : Host-chromosome   Green : Plasmid sequence

License Confirmation for purchasing “TG-Sure Expression (IR/MAR)”

Please accept all the following items:

  1. The use of this product is strictly limited for research purpose only in the organization that   purchaser is belonged.
  2. The purchaser should not make copies and/or derivatives of this product. The purchaser should not transfer and/or resell copies and/or derivatives of this product to any third parties.
  3. The right of intellectual property of the results which is obtained in a study using this product belongs to each researcher.
  4. Use of the product and the results (i.e. expression cell and protein) for any commercial purpose requires a commercial license agreement between TransGenic Inc. and the purchaser.

Please download and print the License Confirmation form (PDF file), after filling in for required matters, and then fax to Cosmo Bio Co., Ltd. before you place the order.

Download the form
References

1. N.Shimizu et al. Cancer Res 61, 6987-6990, 2001
2. N.Shimizu et al. Cancer Res 63, 5281-5290, 2003
3. N.Shimizu et al. Exp Cell Res 302(2), 233-243, 2005
4. N.Shimizu et al. Nucleic Acids Res 33(19), 6296-6307, 2005
5. T.Hashidume et al. J Cell Biochem 101, 552-565, 2007

Product List

Product Name Cat# Quantity Price

TG-Sure Expression (IR/MAR) DataSheet ,

KAL-IR-MAR-DNA01

10 UG ¥112,500
$1500

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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