Transfection of Chinese Hamster Ovary Cells with Beta-Galactosidase Plasmid

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.

Resource Type
Video (49)
Image (439)
Animation (126)
Protocol (38)
Tags
Cell Biology (248)
Genetics (78)
Humans (363)
Environment (270)
Diversity (106)
Teaching (344)

View / Comment
Enlarged view
View / Comment
Labeled View
View / Comment
Enlarged view
View / Comment
Labeled View