Epithelial Regeneration Laboratory

1. Immunohistochemistry.

This technique allows us to look at the cellular localization of different molecules. When fluorescent antibodies are used, cellular localization can be examined using confocal microscopy.

2. Organ Culture of Gut Explants

Segments of gut can be placed in culture. This permits the analysis of the effects of growth factors and specific inhibitors on the differentiation of intestinal epithelial cells.

3. Electroporation

Electroporation is a method for introducing specific gene constructs into the epithelial cell layer of intestinal segments in organ culture. DNA is placed within the lumen and a small electric shock introduces DNA into the cells. This allows the consequences of expression of specific genes on the biology of intestinal epithelial cells to be examined.

Electroporation technique (A) DNA is injected into the lumen and electrical pulses are applied across the gut segment introducing DNA into one side of the epithelium.

(B) Explant electroporated with EF1aEGFP showing many positive cells only on one side of the epithelium. (C) Confocal optical section of epithelium. (D) Transverse section showing EGFP-positive cells.

4. Visualisation of GFP-fusion proteins.

Genes of interest are fused to the green fluorescent protein (GFP) gene, producing a protein that is tagged by GFP. GFP is a protein originally isolated from jellyfish that is fluorescent when exposed to blue light. When these constructs are introduced into the epithelial cell layer by electroporation, the cellular localization of the protein can be examined using high-power fluorescence microscopy.

5. Genetically Manipulated Mouse Models

We use intestinal tissue from genetically manipulated transgenic or conditional knockout mice to investigate potential cancer-causing genes. These mice are engineered to contain mutations that mimic those found in human colon cancer.

6. Embryonic Stem Cell Differentiation

We culture mouse embryonic stem cells and introduce constructs expressing genes implicated in intestinal differentiation. The functional consequences of gene expression are examined in differentiation assays and using qPCR.