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EUCOMMTOOLS Experimental Protocols



Summary of high-throughput gene targeting pipeline.


The process begins with manual Vega annotation of gene structures1 followed by computational design of conditional alleles. Optimal designs are manually selected and oligos/BACs are ordered in 96-well format for high-throughput serial recombineering. Intermediate Gateway-adapted vectors are then assembled into final targeting constructs in a 3-part in vitro reaction. Sequence-verified constructs are used in 25-well parallel electroporations of C57BL/6N ES cells and drug-resistant clones are screened by LR-PCR and sequencing to identify correctly targeted ES cells harboring lacZ conditional alleles.


Experimental Protocols