International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

I11 Identification of Novel Target Proteins in Th2-Dominated Allergic Inflammatory Responses in a Mouse Model of Allergic Asthma: Combining cDNA-RDA and Micro-Arrays

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, whereas 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 (10x), Ig-k light chain (4x) and SLPI (10x). 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, we performed an additional round of cDNA-RDA in which fragments from genes which are already known to be differentially expressed were added to the Driver. For Tester=SAL, suppression of these previously identified products was successful, and a much more complex difference product was obtained. For Tester=OVA, suppression was only partially successful because apparently a too low amount of IgG-g fragments, which appeared to be present at exceptionally high levels in the OVA-amplicons, had been added to the Driver. Therefore, the experiment is being repeated with a much larger amount of IgG-g added to the Driver. Also, for a more comprehensive and faster analysis of the DP's obtained in these experiments, micro-array hybridizations will be used. Genes encoding interesting proteins or unknown genes will be selected for further functional studies. The ultimate challenge is to use this knowledge to explore new therapies for the treatment of allergic asthma aimed at the root causes of the disease.


Abstracts * Officers * Bylaws * Application Form * Meeting Calendar * Contact Information * Home * Resources * News and Views * Membership

Base url
Last modified: Saturday, November 3, 2012