Establishment of MDCK Cells with Stable Overexpression of Canine EGFR Protein and Its Effect on Apoptosis and Cell Cycle

doi:10.7668/hbnxb.20194664

Abstract

Abstract:

Epidermal growth factor receptor(EGFR)is excessively expressed in numerous solid tumours,establishing a stable over-expressing canine EGFR protein MDCK cell line will prove beneficial in providing an exceptional 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,following puromycin selection and single-cell cloning.The infectivity efficiency was observed,expressing gene and protein 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 overexpression 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 ligated into linearized expression vectors,generating 8 positive transformants with a size of 738 bp.The obtained lentivirus titer of EGFR gene overexpression was 3.5×10⁸ TU/mL.Transfected MDCK cells exhibited superior fluorescent 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 enhancement 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 stable canine EGFR overexpressing MDCK cell line.Overexpression of canine EGFR stimulated cell division,elevated DNA replication,and concurrently triggered programmed cell death.

Key words: Madin Darby canine kidney(MDCK), Epidermal growth factor receptor(EGFR), Lentiviral vector

YANG Di, HUANG Lingwei, YANG Yawen, QIAO Zilin, WANG Jiamin. Establishment of MDCK Cells with Stable Overexpression of Canine EGFR Protein and Its Effect on Apoptosis and Cell Cycle[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 223-230. doi: 10.7668/hbnxb.20194664.

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Figures/Tables 8

Tab.1 Linearized vector recombination reaction system with EGFR gene amplification product μL

反应体系 Reaction system	阳性对照 Positive control	自连对照 Self-linking control	试验组 Experimental group
双重去离子水ddH₂O	2.5	4.5	3.5
5×CE Ⅱ缓冲液	2.0	2.0	2.0
5×CE Ⅱ Buffer
酶切后的载体DNA	2.5	2.5	2.5
Carrier DNA after digestion
PCR产物片段	2.0	0.0	1.0
PCR product fragment
重组酶Ⅱ Exnase^TX Ⅱ	1.0	1.0	1.0
合计Total	10.0	10.0	10.0

Tab.2 EGFR and reference gene primer sequence

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')
EFGR_Forward	TAATGGAATAGGGATTGGAG
EFGR_Reverse	TAGAGGTAGGGTATGCGTGA
GADPH_Forward	AGTGACACCCACTCTTCCACCT
GADPH_Forward	GTGGTCCAGGAGGCTCTTACTC

Fig.1 The gene zones and transcripts of the canine reference genome ROS_Cfam_1.0,derived from NCBI browsing software

Fig.2 Analysis of GFP fluorescence expression in stably transformed cell line by fluorescence microscopy A.lv-EGFR cells brightfield,40×;B.lv-EGFR cells darkfield,40×;C.lv-nc cells brightfield,40×;D.lv-nc cells darkfield,40×.

Fig.3 Exploration of mRNA and protein expression in stable transfected cell lines A.Relative expression levels of EGFR mRNA in the overexpression cell line(lv-EGFR) and its control(lv-nc);B.Relative expression levels of EGFR protein in the overexpression cell line(lv-EGFR) and its control(lv-nc).Different capital letters indicate the difference is extremely significant (P<0.01).The same as Fig.5—6.

Fig.4 Indirect immunofluorescence method to detect the expression of EGFR protein in the stably transfected cell line

Fig.5 Flow cytometry assessing the impact of EGFR on cellular apoptosis A.Phenoype of apoptosis in lv-nc and lv-EGFR cells;B.Rates of early and late cell apoptosis within each group.Different lowercase letters indicate significant differences(P<0.05).

Fig.6 Fluorescence-activated cell sorting technology examining the influence of EGFR on cellular cycling dynamics A.Fluorescent distribution charts of the cycle stages of lv-nc and lv-EGFR cells;B.Percentage of cells in each group in G0/G1,S,and G2/M phases.

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