Identification of essential and non-essential protein kinases by a fusion PCR method for efficient production of transgenic Trypanosoma brucei.

Manipulation of gene expression has been used to elucidate gene function, explore fundamental biological processes and to identify potential drug targets in Trypanosoma brucei. We show in bloodstream forms that CDC2-related kinase CRK12 (Tb11.01.4130) is essential since transcriptional inactivation in conditional null mutants is lethal but 19 other protein kinases are not essential since null mutants are viable. We did so using efficient methods for the generation of null and conditional null cell lines of T. brucei by approaches that generate transfection constructs with large targeting sequences and which use reliable transfection and selection conditions. These methods, which are described in detail in the supplementary material, employ multiple oligonucleotides and PCR reactions and several transfections but are cost effective and can simultaneously generate 24 transfectants thus shifting the rate limiting experimental steps from the production of cell lines to their analysis.