The full length version of this protocol, authored by Daniel R. Clayburgh and Jerrold R. Turner, Department of Pathology, The University of Chicago, Chicago, IL 60637, USA, is available from Bio-Rad (bulletin 5370).

Introduction

Tight junctions between intestinal epithelial cells are an important component of the permeability barrier separating the potentially harmful contents of the intestinal lumen from the internal milieu. Cultured monolayers of polarized epithelial cell lines, such as MDCK, T84, and Caco-2, are important model systems for the study of tight junction structure and function. The recent development of siRNA technology provides a new method of probing tight junction regulatory pathways in cell models. Unfortunately, the Caco-2 cell line is relatively resistant to transfection, making the use of siRNA technology technically difficult. Caco-2 cells must be transfected in suspension before plating; cells then typically require a week or more of culture to develop functional tight junctions. Since most siRNAs have a half-life within cells of less than 4 days, traditional methods of Caco-2 transfection are not adequate for siRNA-mediated knockdown. Knockdown using siLentFect-mediated transfection of siRNA and high-density plating of Caco-2 cells allowed the early development of tight junctions, permitting the study of tight junction physiology. With this method, we were able to interfere with expression of myosin light chain kinase (MLCK)1, a splice variant of MLCK, and to study the effect on electrophysiology. Abbreviated Methods

Caco-2 cells were plated on permeable supports. A sequence unique to the MLCK1 splice variant was used to design the siRNA. siRNA or nonspecific control siRNA solutions were added to buffer containing siLentFect lipid reagent and allowed to incubate. At the same time, Caco-2 cells were incubated with trypsin. After trypsinization, the cells were resuspended. The washed suspension was added to previously prepared transfection mixes. The cell suspension and siRNA mixes were then plated at high density and cultured for 4 days to allow tight junction assembly and polarization before use in electrophysiology experiments. Quantitation of MLCK1 and MLCK2 mRNA was performed using PCR and Western blotting. Electrical potential differences were measured, and transepithelial resistance was determined using Ohm's law. Results and Discussion

siRNA-mediated knockdown of MLCK1 in Caco-2 cells was measured by semi-quantitative reverse transcription PCR of RNA from cells transfected with either nonspecific control siRNA or MLCK1-specific siRNA (Figure 1A). MLCK1 mRNA content of monolayers transfected with control siRNA was 52% of the total MLCK expressed. MLCK1 mRNA was reduced to 27% of total MLCK mRNA, a 47% reduction, in monolayers transfected with MLCK1-specific siRNA. MLCK2 mRNA content was unchanged by the MLCK1-specific siRNA.

Fig 1. (Click to enlarge)

We confirmed that siRNA reduced expression of MLCK1 by immunoblotting cell lysates (Figure 1B). Only cells*

*Data from Clayburgh D.R., et al., A differentiation-dependent splice variant of myosin light chain kinase. MLCK1, regulates epithelial tight junction permeability, J. Biol. Chem. 279. 55506-55513 (2004). transfected with the specific siRNA showed a significant reduction in MLCK1 protein. This reduction in expression had a significant effect on the tight junction permeability of the Caco-2 monolayers; cells transfected with MLCK1 siRNA exhibited a significant increase in transepithelial resistance compared to those transfected with nonspecific siRNA (Figure 2). The use of siLentFect in conjunction with high-density plating allowed us to measure the effects of knockdown on tight junction physiology and the molecular regulation of intestinal permeability.

Fig 2. (Click to enlarge)

*Data from Clayburgh D.R., et al., A differentiation-dependent splice variant of myosin light chain kinase. MLCK1, regulates epithelial tight junction permeability, J. Biol. Chem. 279. 55506-55513 (2004).