NorCOMM Gene Targeting Vector Construction PipelineGCOE Program Seminar(Global Education Seminar)

In collaboration with the Sanger Institute, we have established a pipeline for high throughput gene targeting vector construction. In order to achieve the simultaneous processing of samples in 96-well format, the pipeline does not involve the standard cut-and-paste approach. We utilize two in vivo recombination systems both of which were originally derived from bacteriophage lambda: Red recombination (a.k.a., recombineering) and Gateway cloning. As the sole source of mouse genomic DNA fragments, we use bacterial artificial chromosome (BAC) clones which have been sorted, annotated and are readily available on request. Four BAC clones are used for each gene to ensure successful vector construction. Using recombineering, we first replace a critical exon along with its surrounding region with a positive/negative selectable marker. This replacement also introduces attR-sites at border of the marker. Second, we use gap repair to extract a large part of the surplus genomic region within the BAC, leaving ~10kb of gene specific sequence. The resulting new plasmid backbone is flanked by two attR-sites. The positive/negative selectable marker and the plasmid backbone of validated constructs are subsequently replaced with the “NorCOMM allele cassette” and a new plasmid backbone containing a negative selection marker DTA, respectively, via a three way Gateway reaction. Successful construction of the targeting vector is confirmed by sequencing of the construct. The Gateway reaction achieves faithful exchange of cassettes on single base level, eliminating the need of sequencing to confirm the large artificially introduced cassettes in the gene targeting vector.