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Transfection of Chinese Hamster Ovary Cells with Beta-Galactosidase Plasmid

  • Author: Patrick J. Cummings 1
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Johns Hopkins University, Baltimore and Rockville, MD, 21218
  • Citation: Patrick J. Cummings. 2007. Transfection of chinese hamster ovary cells with beta-galactosidase plasmid.
  • Publication Date : June 2007
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Introduction



Figure 1 shows a monolayer of Chinese hamster ovary (CHO) cells stained for endogenous beta-galactosidase activity using X-Gal substrate.  Using bright-field microscopy, the light blue stained cells indicative of background endogenous beta-galactosidase activity in CHO cells can be seen.   
 

Figure 2 shows a monolayer of CHO cells that have been transfected with the pSV-beta-galactosidase control plasmid and stained for beta-galactosidase activity.  Using bright-field microscopy, the dark blue stained cells indicative of the expressed beta-galactosidase enzyme activity in CHO cells can be seen.   



Methods



CHO cells were transiently transfected with one microgram of pSV-beta-galactosidase plasmid using Lipofectin transfection reagent (Invitrogen). Following transfection, cells were grown at 37°C in 5% CO2 for 4 days in Ham's F-12 medium containing 5% fetal calf serum, fixed, and stained with X-Gal substrate.



Discussion 



Chinese hamster ovary cells are mammalian cells commonly used in industrial settings to express recombinant proteins for research and therapeutic applications. The pSV-beta-galactosidase control plasmid is a reporter vector used for monitoring transfection efficiencies of mammalian cells. The SV40 early promoter drives transcription of the bacterial lacZ gene, which is translated into the beta-galactosidase enzyme. Beta-galactosidase enzyme is detected by histochemical staining with X-Gal substrate, which is cleaved by the enzyme to produce a blue end product.  

 

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