Ardabil IAU Researchers Present New Method for Efficient Gene Transfer

Saturday, July 25, 2020 - 11:15
Gene Transfer

Researchers of Ardabil Islamic Azad University have succeeded in presenting a new method which could increase the efficiency of gene transfer in E. coli through applying cationic carbon nanotubes.

According to an ISCA report, in this research which has been carried out by Sheyda Jahazi and Hashem Yaghoubi, and its results released in the journal of Tarbiat Modares University, Pathobiology Research, carbon nanotubes were coated using PEI. Then, their ability to gene delivery to E. coli cells was examined.

Based on the research, nanotube- PEI nanoparticles were synthesized by the reaction between amine groups of PEI and carboxyl groups of nanotubes. In order to prepare the appropriate DNA vector for delivering to E. coli cells, the Gus A gene was transferred from pBI121 to PUC18 vector (pUC-Gus).

Nanotube-PEI/DNA complexes were prepared by combining different mass ratios of nanotube-PEI (0.5, 1, and 2 w/w%) with the fixed amount of DNA. To the transformation of E. coli, the appropriate amount of nanotube-PEI/DNA complexes was added to E. coli cells under stirring at 37°C for 7h. The transformation efficiency of E. coli was determined by colony counting on LB agar supplemented with Ampicillin.

Moreover, Gus staining assay was used to confirm the function of the plasmid. Determination of cytotoxicity of nanotube-PEI was performed using MTT assay at 6, 24, and 72 hours intervals at different concentrations of nanotube-PEI (10, 100, and 500μg/ml).

The nanotube-PEI was synthesized successfully. Nanotube- PEI nanoparticles have a great ability to protect DNA from enzymatic digestion. The percentage of E. coli cells viability was decreased by increasing both the concentration of nanotube-PEI nanoparticles and also the duration of incubation.

The results of the agarose gel electrophoresis of plasmid extracted from E. coli and digested using EcoRI enzyme showed that the pUC-Gus plasmid has been successfully transfected by nanotube-PEI nanoparticles to E. coli bacterial cells.

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