title: A new method for simultaneous gene deletion and down-regulation in Brucella melitensis Rev.1.
abstract: In this study, our aim was to integrate an antisense expression cassette in bacterial chromosome for providing a long-term expression down-regulation in a bid to develop a new approach for simultaneous deletion and down-regulation of target genes in bacterial system. Therefore, we were used this approach for simultaneous deletion of the perosamine synthetase (per) gene and down-regulation of the virB1 expression in Brucella melitensis Rev.1. The per gene, which is one of the LPS O-chain coding genes, was replaced by homologous recombination with an antisense virB1 expression cassette together with kanamycin resistance cassette (kan(R)). Deletion of the per gene was characterized by PCR analysis and DNA sequencing. The expression of antisense virB1 cassette was confirmed by RT-PCR. Down-regulation of the virB1 mRNA expression was quantified by real-time RT-PCR using virB1 specific primers relative to the groEL reference gene. The survival rate of mutant strain was evaluated by CFU count in the BALB/c mice. The virB1 mRNA expression was down-regulated on average 10-fold in mutant strain as compared to parental strain. The loss of per gene function and decrease of the virB1 mRNA expression resulted in reduced entry and survival of the mutant Rev.1 strain in BALB/c mice splenocytes. We propose that this method can be used for simultaneous regulation of multiple genes expression.
authors.affiliation: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box: 14115-175, Tehran, Iran. Electronic address: a.saeedinia@modares.ac.ir.
authors.forename: Ali Reza
authors.initials: AR
authors.lastname: Saeedinia
authors.affiliation: Department of Genetics, Science and Biotechnology Research Center, Mallek-Ashtar University of Technology, P.O. Box: 15875-1774, Tehran, Iran. Electronic address: Zeinoddini@mut.ac.ir.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Zeinoddini
authors.affiliation: Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-111, Tehran, Iran. Electronic address: soleim_m@modares.ac.ir.
authors.forename: Masoud
authors.initials: M
authors.lastname: Soleimani
authors.affiliation: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box: 14115-175, Tehran, Iran. Electronic address: sadeghma@modares.ac.ir.
authors.forename: Majid
authors.initials: M
authors.lastname: Sadeghizadeh
mesh_terms: D000818:Animals; D017347:Brucella melitensis / Q000235:genetics*; D002006:Brucellosis / Q000382:microbiology; D004195:Disease Models, Animal; D015536:Down-Regulation; D005260:Female; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D009690:Nucleic Acid Conformation; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics
chemical_list: D016372:RNA, Antisense
keywords: Antisense RNA; B. melitensis Rev.1; Down-regulation; Perosamine synthetase; virB1