Establishment of MDCK Cells with Stable Overexpression of Canine EGFR Protein and Its Effect on Apoptosis and Cell Cycle
Epidermal growth factor receptor(EGFR)is excessively expressed in numerous solid tumours,estab-lishing a stable over-expressing canine EGFR protein MDCK cell line will prove beneficial in providing an excep-tional cellular model and research foundation for the study of canine or human tumour diseases.Firstly,the target fragment was prepared for construction of an overexpression vector of the EGFR gene.It was co-transfected with a lentivirus packaging vector into 293T cells.After 48 hours of transfection,the supernatant was extracted and purified to obtain a lentivirus containing the overexpressed EGFR gene.This virus was then transduced into MDCK cells,fol-lowing puromycin selection and single-cell cloning.The infectivity efficiency was observed,expressing gene and pro-tein levels were detected through RT-qPCR,Western Blot,and indirect immunofluorescence assays,and an EGFR protein overexpression MDCK cell line was established.Using flow cytometry,apoptosis and cell cycle of overexpres-sion and control cell lines were separately determined.The canine EGFR gene was located on chromosome 18 and consisted of 30 exons encoding a transmembrane glycoprotein.Through the reaction system,PCR products were liga-ted into linearized expression vectors,generating 8 positive transformants with a size of 738 bp.The obtained lenti-virus titer of EGFR gene overexpression was 3.5 × 108 TU/mL.Transfected MDCK cells exhibited superior fluores-cent effect was remarkable under fluorescent microscopy,resulting in a significant increase in the expression level of the EGFR gene and its protein within the cells.Indirect immunofluorescence assay revealed a substantial enhance-ment in the expression of EGFR in cells.In contrast,early apoptosis and late apoptosis rates among the transfected cells increased notably,with a clear arrest occurring at the G2/M phase.This research successfully developed a sta-ble canine EGFR overexpressing MDCK cell line.Overexpression of canine EGFR stimulated cell division,elevated DNA replication,and concurrently triggered programmed cell death.
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