WP 10. Validation of targets: in humanised in vivo and human in vitro models

This area of work will be lead by Partner 10 (Zürich) with input from Partner 3 (Berlin-Charité) and partner 6 (AMC Amsterdam)

Based on our experience in characterisation of endogenous retroviral gene sequences in the synovium of patients with RA and our description of novel downstream signalling pathways like L1, p38 d and MMP1, our laboratory is well equipped to elucidate novel pathways by functional genomics.

1) The selection of identified genes suitable for further investigation will require the contribution of all collaborators. The following criteria will be used for selection:

• Quantitative differences (genes highly differentially expressed) in synovial fibroblasts
• Specificity (genes predominantly expressed in RA patients)
• Expression levels (at both, RNA and protein levels)
• Relevance (gene can be linked to the concept of RA) with respect to genes related to cellular activation
• Novelty (gene not linked to RA so far).

2) For quantification of mRNA, SYBR Green real-time PCR will be performed.

Specific primer pairs for each gene will be designed with the Primer Express software. Samples without enzyme in the reverse transcription will be used as a negative control to exclude genomic contamination. Unspecific signals caused by primer dimers can be excluded by dissociation curve analysis, analysis of the reaction products on agarose gels and by no template controls.

3) Those genes, for which the differential expression has been confirmed by real-time PCR, will be further investigated by in situ hybridisation (ISH) and/or immunohistochemistry (IHC). These methods allow visualising the expression patterns of certain genes in vivo. ISH and/or IHC will be performed on paraffin embedded tissue, or if necessary, on frozen sections. Confirmation on protein levels by IHC will be favoured, if antibodies are available. Otherwise ISH will be performed. In addition, immunohistochemical double labelling will be used to further specify the expression on different cell types. For the evaluation of the results from immunostaining, a scoring syste m will be assigned, and digital image analysis will be performed.

4) To test the relevance of the selected and confirmed genes in the SCID mouse co-implantation model, RA synoviocytes as well as normal synovial fibroblasts will be co-implanted with normal human articular cartilage under the renal capsule of SCID mice and analysed for their destructive potential and studied for the expression of the genes of interest. Emphasis will be placed, whether the engrafted RA-SF express the gene products at sites of cartilage invasion. These genes will be detected by using ISH or IHC.

5) Functional analysis of genes, which are expressed at sites of invasion in the SCID mouse model, will be performed. For this approach, normal synovial fibroblasts will be transfected with retroviral constructs encoding for the sequences of interest or mock vectors to study in the SCID mouse model whether these transfected normal cells become invasive.