3385_Ch11 - Chapter 11 Methods for Studying Anoikis Steven...

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11 Chapter Methods for Studying Anoikis Steven M. Frisch Contents I. Introduction II. Assaying the Effects of Genes on Anoikis in Stable Expression Experiments A. Generating Stable Expressions B. Assaying Stable Expressors for Anoikis III. Transient Assays for the Effects of Genes on Anoikis A. β -Galactosidase/DAPI Double-Staining Method B. Keratin 18-Cleavage Transient Assay for Effects of Transgenes on Anoikis IV. Materials References I. Introduction Epithelial cells deprived of normal matrix contact through the appropriate integrin heterodimer undergo apoptosis; this phenomenon has been termed “anoikis,” the ancient Greek word meaning “homelessness.” 1 Anoikis prevents shed epithelial cells from colonizing elsewhere. 2 This process is important for normal development, and its loss can contribute significantly to tumor malignancy (reviewed in Reference3). Assays for the effects of compounds or genes on anoikis are currently under development, several of which are described here. Each has its advantages and disadvantages. The most time-consuming and conventional is stable expression of new genes in epithelial cells, and assaying the resulting cell lines for anoikis relative © 1999 by CRC Press LLC
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to the parental cells; this assay has been used successfully to show the role of certain signaling molecules such as FAK and MEKK-1. 4,5 This chapter will specify the stable gene transfer and apoptosis assay methods that work best in our experience with epithelial cells. In addition, we describe transient assays that are much faster but whose track record is less established. II. Assaying the Effects of Genes on Anoikis in Stable Expression Experiments A. Generating Stable Expressors MDCK cells are a widely used epithelial cell model system. It has two drawbacks, though. First, being a canine cell line limits the choice of antibodies and nucleic acid probes, although many anti-human antibodies or probes work in this species. MDCK cells are also poorly transfectable, prompting the use of retroviral vectors. We mainly use the vector pBABE. 6 This vector drives expression of the insert through the viral LTR enhancer and has an internal SV40 promoter to drive the expression of a puromycin-resistance gene for selection. Following subcloning of the gene of interest into pBABE (usually in a FLAG-, myc-, or HA-epitope-tagged form), the retrovirus vector is transfected by the standard calcium phosphate method into the amphotropic packaging cell line φ NX, 7 which is much more efficient than previous packaging cell lines. Two to three days after transfection, the viral super- natant is removed, cleared by centrifugation (3000 × g for 10 min), polybrene is added to give 4μg/ml final concentration, and it is applied to a subconfluent mono- layer of MDCK cells. The MDCK cells can either be on tissue culture plastic, or, for higher efficiency, on permeable cell culture inserts of 25mm diameter and 3.0 micron pore size (Falcon), which fit into 35mm wells. For the latter, the viral
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This note was uploaded on 05/06/2010 for the course MECH. 28197 taught by Professor Dr.shafii during the Spring '10 term at Sharif University of Technology.

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3385_Ch11 - Chapter 11 Methods for Studying Anoikis Steven...

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