Authors : Payman Zare , Mohammad Tabatabaei , Ghasem Yousefbeygi and Ahmad Reza Jabbari
Abstract: An aroA mutant of a local strain of Pasteurella multocida A: 1 was produced using Red recombinase and PCR product. A pair of primers (aroAPmF and aroAPmR) were used that contained sequences homologous to regions adjacent to the gene to be inactivated followed by sequence of a flanking FRT site. A PCR process with these primers and the template plasmid pKD4 containing a kanamycin resistance gene with FRT (FLP recognition site) flanks was performed and the purified product (~1.6 kb) was transformed into the wild type strain which had already been transformed with pKD 46, a plasmid containing Red recombinase gene. The transformed product replaced the original gene by homologous recombination. This method does not need cloning or sequencing of the gene, any restriction enzyme cuts or special shuttle or suicide vectors. The mutant strain was selected on media containing kanamycin and verified with PCR using both pairs of primers (AroAPmF and AroAPmR, AroA1F and AroA2R), then tested for attenuation and immunity induction by challenge in chickens, as mutation in the aroA gene creates dependency for growth on aromatic compounds that are not available in host tissues and the aroA mutants of P. multocida would provide good protection against challenge in chickens.
Payman Zare , Mohammad Tabatabaei , Ghasem Yousefbeygi and Ahmad Reza Jabbari , 2008. Using Red Recombinase and PCR Product for One-Step in-Frame Inactivation of aroA Gene in Pasteurella multocida A: 1 and Evaluation of Pathogenicity and Immunogenicity. Journal of Animal and Veterinary Advances, 7: 1155-1159.