Report of the visit of S. Metzdorf of the VFA to the RIKILT (GMOCARE-SAFOTEST)
Regarding the visit of Stine Broeng Metzdorff from the Danish Veterinary and Food Administration (VFA), to RIKILT, Department of Food Safety and Health, the Netherlands in July 2001.
One of the main purposes in the GMOCARE RTD project is to employ a holistic approach in the analysis of transgenic plants. Thus, the same transgenic plants, as well as relevant control lines, are subject to a hole range of different molecular and biochemical analysis, to demonstrate whether the GM crops/plants do or do not differ substantially from their classical bred cultivars.
In one of the work packages of the GMOCARE project (WP5), the objectives is to utilise the DNA micro-arrays technology as a comprehensive tool to identify genes responding to the genetic modifications made, being either expected in relation to the transgenic modification or unintended effects (i.e. metabolic perturbations). One of the plants that are to be investigated is genetically modified Arabidopsis thaliana plants, which also serves as a model plant in the project. In sub-work package 5.2 the main objective is, in collaboration with RIKILT and VFA, the construction of a cDNA micro-array containing a range of Arabidopsis cDNAs. In this collaboration VFA is responsible for the isolation and construction of the cDNA library, whereas RIKILT is dealing with the construction of the micro-array as well as the hybridisation experiments. In another EU-financed RTD project, SAFOTEST, both RIKILT and VFA are participants and also here RIKILT participate doing cDNA micro-array (rat cDNA).
With the aim of enhance the collaboration between the GMOCARE and the SAFOTEST project, but also between RIKILT and VFA, Ph-D student Stine Broeng Metzdorff (SBM), VFA, visited for a period of 3 weeks RIKILT in order to work with both Esther Kok and Ad Peijnenburg, working on the GMOCARE and SAFOTEST projects, respectively.
During the 3 weeks at RIKILT, SBM participated in the following procedures in order to fabricate the micro-array chip. In the first week, 16 x 96 wells with a total of 1536 clones including controls were amplified by PCR with 5hexamine-modified primers (1F-mod and 3R-mod). Subsequently, the PCR amplifications were analysed on agarose gels in order to check if the amplifications were performed correctly and to register any empty clones. The clones used for the PCR amplification contained Arabidopsis cDNA fragments obtained from subtractive PCR performed in Denmark by SBM. During the second week, the PCR products were purified with Sephadex G-50 in Millipore Multiscreen-HV plates. The purified PCR products were then precipitated with NaAc + isopropanol, centrifugated and washed, and after drying with speedvac, the pellets were dissolved. In the third, and last week of the visit, the spotting layout were designed and each purified PCR product were then transferred from 16 x 96-wells plates to 4 x 384-wells plate, which are the type of plates that are used during the spotting procedure. Finally, the PCR-products were spotted on silylated glass slides with the PixSys 7500 arrayer, Cortesian Technologies, with 4 micro spotting pins performing contact printing. 50 slides/arrays were produced and after spotting, the arrays where stored in a box for drying. In a future visit to RIKILT, SBM will participate in the hybridisation of the arrays with totRNA isolated from transgenic Arabidopsis plants and also participate in analysing the results obtained from the hybridisation.
After 3 weeks the work was finished as planned and the aims for visiting RIKILT was carried out successfully, both concerning the development of a closer collaboration between the laboratories, but also regarding the good opportunity for SBM, as well as VFA, to obtain some knowledge on the field of the micro-array technique.