This chapter reviews the methods employed to transfect and express foreign genes into epithelial cells. It also determines the polarized surface expression of both endogenous and exogenous plasma membrane proteins. The polarized distribution of surface proteins between the apical and the basolateral surfaces of epithelial cells is the basis of their vectorial function. Retroviral promoters, specifically Moloney murine leukemia virus (MuLV) and Rous sarcoma virus long terminal repeats (LTR) have resulted in high levels of expression of exogenous plasma membrane proteins. The neomycin-resistance marker coupled with selection with G418 has been very useful for the selection of stable transfectants. Polarity can be expressed as a ratio between the amounts of a given molecule in apical and basolateral plasma membrane domains. An important consideration in the analysis of this ratio is the fact that the area of the apical domain is usually a fraction of the area of the basolateral domain. It is important to express polarity as a density ratio—that is, a ratio between the amounts of the molecule per unit surface area in each surface domain. Various methods to estimate the surface polarity of a molecule in expressed epithelial cells include isotopic uptake or binding, electrophysiologic procedures, postsection colloidal gold immunocytochernistry, and EM immunocytochemistry.
ASJC Scopus subject areas
- Cell Biology