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. 2023 Jul;45(7):861-871.
doi: 10.1007/s10529-023-03387-7. Epub 2023 May 11.

Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system

Maryam Zare  1   2 , Ali Farhadi  1   2 , Farahnaz Zare  1 , Gholamreza Rafiei Dehbidi  1 , Farzaneh Zarghampoor  1 , Mohammad Karimi Baba Ahmadi  3 , Abbas Behzad Behbahani  4
Affiliations

Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system

Maryam Zare et al. Biotechnol Lett. 2023 Jul.

Abstract

Purpose: Despite advances in gene therapy, the lack of safe and efficient gene delivery systems limited the clinical effectiveness of gene therapy. Due to the inherent potential of bacteria, they can be considered as a good option for the gene transfer system. This study aimed to create a genetically engineered bacterium capable of entering epithelial cells and transferring its genetic cargo to the cell's cytoplasm, eventually expressing the gene of interest in the cell.

Methods: The invasin (inv) gene from Yersinia pseudotuberculosis and the listeriolysin (hlyA) gene from Listeria monocytogenes were isolated by PCR assay and inserted into a pACYCDuet-1 vector. The recombinant plasmid was then transformed into E. coli strain BL21. Subsequently, pEGFP-C1 plasmids containing a CMV promoter were transformed into the engineered bacteria. Finally, the engineered bacteria containing the reporter genes were incubated with the HeLa and LNCaP cell lines. Fluorescence microscopy, flow cytometry, and TEM were used to monitor bacterial entry into the cells and gene expression. We used native E. coli strain BL21 as a control.

Results: A fluorescence microscope showed that, in contrast to the control group, the manipulated E. coli were able to penetrate the cells and transport the plasmid pEGFP-C1 to the target cells. Flow cytometry also showed fluorescence intensity of 54.7% in HeLa cells and 71% in LNCaP cells, respectively. In addition, electron micrographs revealed the presence of bacteria in the cell endosomes and in the cytoplasm of the cells.

Conclusion: This study shows that genetically engineered E. coli can enter cells, transport cargo into cells, and induce gene expression in the target cell. In addition, flow cytometry shows that the gene transfer efficiency was sufficient for protein expression.

Keywords: Endosomes; Escherichia coli; Flow cytometry; Gene transfer techniques; HeLa cells; Yersinia pseudotuberculosis.

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