Peter C. Groot, Wouter Gubbels, Jeroen B. Van Bergenhenegouwen, Frans P. Nijkamp, and Antoon J.M. Van Oosterhout
Dept of Pharmacology and Pathophysiology, Utrecht Institute of Pharmaceutical Sciences, Universiteit Utrecht, The Netherlands.
We have developed a model in the mouse with immunologic and pathophysiologic features reminiscent of allergic asthma such as antigen-specific IgE, airway eosinophilia and hyperresponsiveness to bronchoconstrictor stimuli. To unravel the regulatory mechanisms involved in the pathways leading to the Th2-dominated allergic inflammatory responses and to identify novel potential drug targets, we used this model to perform expression studies using Representational Difference Analysis of cDNA's (cDNA-RDA). Lung draining lymph-nodes from ovalbumine (OVA) or saline (SAL, control) challenged, OVA sensitized mice were isolated and compared by cDNA-RDA to identify genes upregulated (Driver=SAL, Tester=OVA) or downregulated (Driver=OVA, Tester=SAL) after OVA-challenge. After 2 rounds of subtractive hybridization, difference products (DP2's) of limited complexity were obtained. In the subtraction with Tester=SAL, one major product, Cyp2f2, was obtained, as well as about 30 fragments derived from other genes. Cyp2f2 was downregulated at least 10-fold, wheareas 14 other genes were downregulated 1.5-4 times. In Tester=OVA, 3 main products comprised about 90% of the difference product obtained, derived from three genes which were strongly upregulated after OVA-challenge, IgG-g (³10x), Ig-k light chain (³4x) and SLPI (³10x). This result is in agreement with the observation that cDNA-RDA yields only a small number of cDNA-fragments, derived from differentially expressed genes, even when a large number of genes are differentially expressed, and that most of these fragments are derived from genes expressed at high(er) levels (Groot and Van Oost, NAR 26, 4476-4481, 1998). To obtain larger numbers of fragments, derived from genes expressed at lower levels, additional rounds of cDNA-RDA are being performed, in which fragments from genes which are already known to be differentially expressed are added to the Driver. For a more comprehensive and faster analysis, cDNA-RDA will be combined with micro-array technology. To this end, several hundred cloned difference products have been spotted on micro-arrays. Hybridization with Tester- and Driver-amplicons will reveal the level of differential expression of each difference product. Hybridizations with (mixes of) previously sequenced difference products will reveal new (non-hybridizing) clones, allowing the analysis of many hundred clones in a straightforward fashion. The advantages of combining cDNA-RDA and micro-arrays are the possibilities to enrich both target and probe for differentially expressed sequences and the fact that micro-arrays with only a limited and therefore manageable number of clones (£ 1000) are sufficient for a comprehensive analysis.