J Vet Sci.  2014 Mar;15(1):111-115. 10.4142/jvs.2014.15.1.111.

Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells

• An L
• Liu S
• Zhang W
• Zhang Y
• Huang Y
• Hu X
• Chen S
• Zhao S

• KMID: 1737616
• DOI: http://doi.org/10.4142/jvs.2014.15.1.111

Abstract

Molecular mechanisms underlying the effects of Fyn on cell morphology, pseudopodium movement, and cell migration were investigated. The Fyn gene was subcloned into pEGFP-N1 to produce pEGFP-N1-Fyn. Chinese hamster ovary (CHO) cells were transfected with pEGFP-N1-Fyn. The expression of Fyn mRNA and proteins was monitored by reverse transcription-PCR and Western blotting. Additionally, transfected cells were stained with 4',6-diamidino-2-phenylindole and a series of time-lapse images was taken. Sequences of the recombinant plasmids pMD18-T-Fyn and pEGFP-N1-Fyn were confirmed by sequence identification using National Center for Biotechnology Information in USA, and Fyn expression was detected by RT-PCR and Western blotting. The morphology of CHO cells transfected with the recombinant vector was significantly altered. Fyn expression induced filopodia and lamellipodia formation. Based on these results, we concluded that overexpression of mouse Fyn induces the formation of filopodia and lamellipodia in CHO cells, and promotes cell movement.

Keyword

filopodia; Fyn; lamellipodia; time-lapse

MeSH Terms

Animals
Blotting, Western
CHO Cells
Cricetinae
Cricetulus
Genetic Vectors
Green Fluorescent Proteins/genetics
Mice
Proto-Oncogene Proteins c-fyn/genetics/*metabolism
Pseudopodia/*metabolism
Reverse Transcriptase Polymerase Chain Reaction
Time-Lapse Imaging
Transfection
Green Fluorescent Proteins
Proto-Oncogene Proteins c-fyn

Figure

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  Fig. 1 Identification of the recombinant plasmid. (A) Detection of the housekeeping gene GAPDH. (B) Identification of the target gene by RT-PCR. (C) PCR product of the Fyn gene was subcloned into pMD18-T-Fyn and pEGFP-N1-Fyn. (D) Identification of pMD18-T-Fyn fragments produced by restriction enzyme digestion with EcoR I and Sma I. (E) Identification of pEGFP-N1-Fyn fragments generated by restriction enzyme digestion with EcoR I and Sma I. Lane 1, RT-PCR GAPDH product; Lane 2, negative control; Lane 3, RT-PCR Fyn product from brain; Lane 4, positive control; Lane 5, PCR product of pMD18-T-Fyn; Lane 6, PCR product of pEGFP-N1-Fyn; Lanes 7 and 9, positive control; Lanes 8 and 10, recombinant plasmid identification by digestion with EcoR I and Sma I (pMD18-T-Fyn, 2700 bp; pEGFP-N1, 4700 bp; Fyn, 1611 bp); Lane M, DNA marker.

• 

  Fig. 2 Overexpression of pEGFP-N1-Fyn in CHO cells. (A) Detection of Fyn mRNA in CHO cells by RT-PCR. (B) Expression of Fyn protein in CHO cells identified by Western blotting. (C) Immunohistochemical staining and effects of Fyn expression on cell morphology. Fyn induced filopodia and lamellipodia formation in CHO cells. Lanes 1, 2, and 3 were the blank group (dealt with DMEM/F12K), control group (transfected with pEGFP-N1), and Fyn group (transfected with pEGFP-N1-Fyn), respectively. Magnified fields are labeled as a and b. Scale bars = 10 µm.

• 

  Fig. 3 Movement of a transfected CHO cell observed in a time-lapse series of images. Time-lapse confocal microscopy images show the dynamics of pseudopodium change. The 22 figures are a time-lapse series showing pseudopodium formation 12 h after transfection. Asterisks indicate the real-time location of the pseudopodium. Cells were photographed and cultured within a live cell station. Scale bars = 10 µm.

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