One of the strategies for the construction of live vaccine candidates is through the generation of genetically defined isogenic strains, containing single or multiple mutations in target-specific genes generated by allelic exchange. This approach allows to produce rational attenuation of or, alternatively, sequence-specific modifications to produce variants of antigenic molecules or change their expression levels. Genetic tools amenable for their use in mycobacterial strains have allowed the identification and validation of potential targets for the diagnosis, prevention, and treatment of tuberculosis. However, the genetic manipulation of Mycobacterium tuberculosis and other slow-growing strains such as Mycobacterium bovis BCG has been delayed by various factors related to their physiology and cell wall characteristics. Notwithstanding the foregoing, the high frequency of illegitimate recombination and the availability of few antibiotic selection markers limit the feasibility of genetic manipulation of mycobacterial strains. This chapter describes a protocol for the generation of defined mutants using recombination tools in an inducible recombination system driven by mycobacterial Che9c phage RecET proteins, originally developed in Dr. Graham Hatfull's group, combined with linearized recombination substrates containing flanking sequences of a locus of interest and an antibiotic resistance gene. These recombination substrates contain sites for removal of antibiotics selection markers. This system allows to make marked and unmarked mutations by homologous recombination in a single step as a result of a double crossover between the homologous regions on the genome and the allelic exchange substrate. In addition, this genetic tool used for engineering mycobacterial genomes performs with lower rates of illegitimate recombination and take on average less time to create knock-out (KO) mutant compared with other techniques.
Keywords: Gene replacement; Homologous recombination; Mutation; Recombineering; Vaccine.
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