Cloning of ORFs - genes and splice variants

S. Bechtel, D. Heubner, K. Köhrer, J. Lauber, S. Wiemann

The protein coding regions are PCR amplified based on gene models. Products are cloned into Gateway entry vectors that allow for the shuttling of the ORFs into a variety of expression vectors (Wiemann et al., 2004, Simpson et al., 2000). All ORFs cloned in entry vectors are sequence verified prior to their exploitation in functional gene analysis.

More than 1.200 ORFs have been cloned thus far in entry clones. Over 4.000 expression clones have been generated from these entry clones. Additional to their use in SMP-Cell, the ORF-resource is also utilized in other projects, e.g. to express the proteins in E.coli for the production of protein arrays.

The entry clones generated within SMP-Cell are part of the ORFeome resource of the international ORFeome Collaboration (http://www.orfeomecollaboration.org)

Further reading (http://www.smp-cell.org/Files/Reports/SMP-Cell_ORFCloning.pdf)