title: A lacZ Reporter-Based Strategy for Rapid Expression Analysis and Target Validation of Mycobacterium tuberculosis Latent Infection Genes.
abstract: We report a novel lacZ fusion vector and demonstrate its utility for expression analysis of genes associated with Mycobacterium tuberculosis latent infection. The vector contains E. coli (oriE) and mycobacterial (oriM) origins of replication, a kanamycin resistance gene (Km(r)) as selection marker, and a lacZ reporter gene in fusion with MCS for cloning of upstream regulatory sequence of the desired genes. β-galactosidase activity of the vector was standardized for expression analysis under latent mycobacterial conditions using Phsp60, a constitutive mycobacterial promoter, utilizing Mycobacterium smegmatis as model organism. Validation of the vector was done by cloning and expression analysis of PhspX (alpha crystalline) and Picl (isocitrate lyase), promoters from two of the genes shown to be involved in M. tuberculosis persistence. Both genes showed appreciable levels of β-galactosidase expression under hypoxia-induced persistent conditions in comparison to their actively replicating state. Expression analysis of a set of hypothetical genes was also done, of which Rv0628c showed increased expression under persistent conditions. The reported fusion vector and the strategy can be effectively used for short listing and validation of drug targets deduced from various non-conclusive approaches such as bioinformatics and microarray analysis against latent/persistent form of mycobacterial infection.
authors.affiliation: Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, 173234, India.
authors.forename: Shivani
authors.initials: S
authors.lastname: Sood
authors.affiliation: Division of Microbiology, Central Drug Research Institute, Lucknow, Uttar Pradesh, 226001, India.
authors.forename: Satinder
authors.initials: S
authors.lastname: Kaur
authors.affiliation: Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, 173234, India. juit.rahul@gmail.com.
authors.forename: Rahul
authors.initials: R
authors.lastname: Shrivastava
mesh_terms: D019677:Artificial Gene Fusion; D018834:Chaperonin 60 / Q000235:genetics; D003001:Cloning, Molecular; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D005798:Genes, Bacterial; D005822:Genetic Vectors; D055985:Latent Tuberculosis / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018741:Replication Origin; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
chemical_list: D018834:Chaperonin 60; D001616:beta-Galactosidase