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

Aspartyl/asparaginyl β-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfection, the obtained cells normally contain a range of cells expressing the different levels of the target of interest. In this paper, we report on our simple innovative approach in the selection of best-transfected cells with the highest expression of ASPH using subclone selection, quantitative real-time polymerase chain reaction, and gradual increment of hygromycin concentration.

To achieve this goal, human embryonic kidney (HEK 293T) cells were transfected with an ASPH-bearing pcDNA3.1/Hygro(+) vector. During antibiotic selection, single accumulations of the resistant cells were separately cultured and the ASPH mRNA levels of each flask were evaluated. The best subclones were treated with a gradually increasing amount of hygromycin. The ASPH protein expression of the obtained cells was finally evaluated using flow cytometry and immunocytochemistry.

The results showed that different selected subclones expressed different levels of ASPH. Furthermore, the gradual increment of hygromycin (up to 400mg/mL) improved the expression of ASPH. The best relative fold change in mRNA levels was 57.59 ± 4.11. Approximately 90.2% of HEK^ASPH cells overexpressed ASPH on their surface.

The experiments indicated that we have successfully constructed and evaluated a recombinant human cell line overexpressing ASPH on the surface. Moreover, our innovative selection approach provided an effective procedure for enriching highly expressing recombinant cells.