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

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 expressing α4 integrin 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 vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable truncated TITGA-4 expressing cells. Chromosomal DNA of truncated TITGA-4-transfected cells was extracted and the presence of truncated TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated 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 truncated 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.

An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase

“…We also used qRT-PCR assay as the initial monitoring approach which is substantially less expensive than flow cytometry and second fluorescence selectable reporters. Also, our findings are in agreement with other studies on the effect of increasing hygromycin concentrations, which suggest that higher doses of antibiotics would eliminate less resistant cells ( 16 23 24 ).…”

“…Other than antibiotic selection, some studies use no additional strategies to select higher expression cells. They collect all the antibiotic-resistant cells together in a single flask ( 16 23 24 ). In this traditional process of transfection and selection, the remained cells usually consist of various cells with different protein expression levels.…”

“…The recombinant cells could be used in aptamer selection via the cell-based selection procedure. Moreover, the cells could be further used in monoclonal and polyclonal antibody production ( 16 ). Meantime, the expressed recombinant ASPH could be easily purified for further advanced studies.…”

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

“…Various elements are important to produce demyelinated plaques, including autoantibodies, cytokines, and activated autoreactive CD4+ T cells, which play the most crucial role in this process. 5 The migration of autoreactive immune cells through the blood-brain barrier, is considered as the pathologic hallmark of MS. 6 Based on the importance of α4 integrin in MS pathogenesis, natalizumab (Tysabri), a blocking antibody to α4 integrin was approved for treatment of patients with relapsing MS. [7][8][9] There are some novel therapeutic tools that are capable of target therapy in different context. [10][11][12][13] Aptamers as one of those therapeutic tools are single-stranded DNA (ssDNA) or RNA molecules that are typically less than 100-mer, which have the ability to bind to a wide range of targets, with high affinity and specificity.…”

“…The migration of autoreactive immune cells through the blood‐brain barrier, is considered as the pathologic hallmark of MS . Based on the importance of α4 integrin in MS pathogenesis, natalizumab (Tysabri), a blocking antibody to α4 integrin was approved for treatment of patients with relapsing MS …”