Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin

Azam Fatahi, Ilnaz Rahimmanesh, Mina Mirian, Fattah Rohani, Maryam Boshtam, Azam Gheibi, Laleh Shariati, Hossein Khanahmad, Shirin Kouhpayeh

Abstract


Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn’s disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative α4 integrin expressing cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (TITGA-4) expression. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable TITGA-4 expressing cells. Chromosomal DNA of TITGA-4-transfected cells was extracted and the presence of TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of TITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of TITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin.

 


Keywords


HEK-293T; Integrin α4; ITGA4; Multiple sclerosis.

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