For the purpose of generating a Trypanosoma brucei AnTat 1.1 cell line deprived of AK3, the authors used a double knockout approach. Nucleofections were carried out using the Amaxa Nucleofector (Lonza)(Burkard et al MBP2007) and clonal populations were obtained with sub-cloning by limiting dilution. Generation of the ?ak3 cell line was accomplished by 2 successive rounds of Nucleofection to replace both alleles of endogenous AK3 in WT with BLA and PURO genes. For the purpose of generating an add-back cell line (AB), the AK3::Ty1 gene was synthesized and integrated into the pHD430 plasmid by Genecust Europe to generate the pHD430AK3::Ty1 plasmid. This plasmid was linearized with the NotI restriction enzyme prior to Nucleofection for integration into the ribosomal intergenic spacer in the ?ak3 cell line. An additional round of nucleofection was carried out on clonal AB cell line with the pHD360 vector that was targeted to the tubulin locus to ensure expression of tet-repressor, hence rendering the production of the AK3::Ty1 protein tetracycline-inducible. As the generated cell lines were to be used for tsetse infection experiments, only one clonal cell line from each was chosen for further analyses. Lonza summary: The authors managed to knockdown the AK3, a gene encoding the flagellar arginine kinase, in Trypanosoma brucei. They used a successive double Nucleofection approach to stably impair both AK3 alleles through DNA recombination and different selection markers. The motility and infectivity of the modified parasites was impaired in its tsetse fly vector, and this phenotype was revertable by overexpression of AK3. The authors conclude that AK3 might play an important role in the parasite Trypanosoma brucei infection of tsetse fly.