title: Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation.
abstract: Haemophilus parasuis, belonging to the family Pasteurellaceae, is the causative agent of Glässer's disease leading to serious economic losses. In this study, a successive markerless mutation system for H. parasuis using two sequential steps of natural transformation was developed. By the first homologous recombination, the target genes were replaced by a cassette carrying kanamycin resistance gene and sacB (which confers sensitivity to sucrose) gene using kanamycin selection, followed by the second reconstruction to remove the selection cassette, with application of sucrose to further screen unmarked mutants. To improve DNA transformation frequency, several parameters have been analyzed further in this work. With this method, two unmarked deletions in one strain have been generated successfully. It is demonstrated that this system can be employed to construct multi-gene scarless deletions, which is of great help for developing live attenuated vaccines for H. parasuis.
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Luhua
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Ke
authors.initials: K
authors.lastname: Dai
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Xintian
authors.initials: X
authors.lastname: Wen
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Rui
authors.initials: R
authors.lastname: Wu
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Huang
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Jin
authors.initials: J
authors.lastname: Jin
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Kui
authors.initials: K
authors.lastname: Xu
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Qigui
authors.initials: Q
authors.lastname: Yan
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Ma
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Porcine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, PR China.
authors.forename: Sanjie
authors.initials: S
authors.lastname: Cao
mesh_terms: D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D005819:Genetic Markers; D044137:Haemophilus parasuis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D014170:Transformation, Genetic
chemical_list: D005819:Genetic Markers; D004247:DNA