Genetic knockdown system for Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that is responsible for the highest number of reported cases of sexually transmitted infections. Until recently Chlamydia has been genetically intractable, thereby limiting genetic approaches. Unfortunately, gene inactivation by TargeTron or antibiotic cassette insertion can result in polar effects of neighboring genes making it difficult to study the genes within operons. This study focuses on developing a novel knockdown strategy by expressing the reverse complement specific Chlamydia genes on a shuttle plasmid. Once cloned, the plasmids are transformed back into Chlamydia and the genes expressed in trans will be transcribed and bind the RNA of the corresponding gene producing double stranded RNA which is degraded. This method will allow us to look at individual genes in an operon without the polar effects of mutations. This strategy is being used on an operon containing 6 genes. After the reverse complement of each gene is expressed, the decreased expression of the target gene will be assessed by reverse transcription PCR. These experiments are the first to utilize a gene specific knockdown strategy in Chlamydia