Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC


List of Abstracts * Speakers * Organizers * Authors * Original Announcement


Systematic Cloning of Open Reading Frames into Expression Systems

Stephanie Bechtel, Anny Duda, Kerstin Hettler, Detlev Bannasch, Alex Mehrle, Annemarie Poustka, and Stefan Wiemann
German Cancer Research Center (DKFZ), Dept. Molecular Genome Analysis (H0600), Im Neuenheimer Feld 350, 69120 Heidelberg Germany
telephone: +49 6221 56 4783
fax: +49 6221 56 4782
email: s.bechtel@dkfz.de

The large-scale cDNA sequencing approaches of the German cDNA Consortium within the last 5 years have resulted in the generation and identification of approximately 1000 novel full-length human cDNAs encoding proteins of unknown function. To allow for their functional characterization, a large scale systematic cloning technology applied in a high-throughput manner is essential. To achieve this, we use the Gateway system from Invitrogen, which is based on cloning by site-specific recombination. We systematically amplify the ORFs by 2-step-PCR. The PCR products are cloned by recombination into an entry vector in a one-step reaction (BP reaction) and resulting entry clones are sequenced. Our pool of confirmed entry clones corresponds currently to about 600 ORFs. For functional analysis, the ORFs can be shuttled from one entry clone into any Gateway compatible expression vector in a single step reaction (LR reaction), again by recombination, e.g. for eukaryotic or prokaryotic protein expression in a native form or as fusion proteins. We currently clone the ORFs without a native stop-codon to enable the generation of both N- and C-terminal protein fusions. In the future, we intend to additionally create entry clones containing ORFs with a terminator triplet to express the proteins in “real” native form. This also offers the opportunity to make the cloning procedure independent of destination vector reading frames. In this way, we accommodate the expanding number of destination vectors which are used for comprehensive functional proteomic assays which are currently under development in our group.

For systematic subcellular localization studies of the novel proteins, the ORFs are subcloned into Gateway compatible EYFP and ECFP expression vectors for generation of C-terminal yellow fluorescent and N-terminal cyan fluorescent fusion proteins. These studies are already established in cooperation with the group of Rainer Pepperkok at the EMBL, Heidelberg. In order to allow monitoring of the whole clone construction process all data are entered into a database together with the results of GFP fusion protein localizations which are entered using a web-interface. The localization data are accessible via the internet (http://www.dkfz.de/LIFEdb/).



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