Recombineering of Human TNF BAC

The human TNF BAC clone (RP11-184F16; obtained from Life Technologies, UK) was purified using a Machery-Nagel Nucleobond BAC100 kit and used to transform electro-competent SW102 E. coli (a kind gift from Neal Copeland; Houston Methodist Research Institute, Houston, TX, USA). SW102-TNF positive clones were confirmed by pulsed-field gel electrophoresis analysis. Recombineering was performed using seamLess GalK mediated selection/counter selection strategy, as described previously^{26 (link),48 (link)}. In brief, two targeting cassettes designed to excise the first coding exon of the TNF gene were generated to first incorporate the GalK gene within the BAC, positive recombinants derived by selection on minimal media containing galactose, followed by the removal of GalK and integration of the luciferase-polyA coding sequence from pGL3Basic (Promega, UK) and counter selection using 2-deoxy-galactose and glycerol. Correctly modified colonies were identified by PCR screening and pulsed-field gel electrophoresis.