Gene Expression ServiceServices

• Conceptual design of targeting constructs or transgenic constructs
• Development of Southern screening-strategies for targeted ES cells
• General help in cloning and recombineering technologies
• Design and assembly of targeting vectors (knock-out, conditional knock-out, knock-in targeting constructs) (assembly of a prototypical cKO targeting vector is displayed in the figure below)
• Design and assembly of transgenesis vectors (compound transgenic vectors, BAC transgenic constructs)
• Cloning and assembly of custom-made cassettes
• Maintenance of bacterial stocks and cassette collection for all intermediates and readily assembled constructs available to EMBL researchers  

Schematic representation of an assembly process for cKO targeting constructs. The first reaction involves retrieval of a genomic fragment comprising the to be targeted exon plus homology arms into a plasmid backbone (Step 1). Introduced unique RENs for later linearization are indicated by asterisks. Next, a LoxP-flanked prokaryotic Kanamycin selection cassette is inserted at a position of choice, usually upstream of the splice branch site (Step 2). Using bacterial Cre-expression, the selection cassette and the second LoxP site are excised, leaving behind the intact single LoxP site (Step 3). The last reaction involves insertion of the second LoxP site and a dual pro- and eukaryotic Kanamycin/Neomycin selection cassette (Step 4).