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Article: A method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy
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TitleA method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy
 
AuthorsYu, B1
Yang, M1 2
Wong, HYB1
Watt, RM3
Song, E2
Zheng, BJ1
Yuen, KY1
Huang, JD1
 
KeywordsRecombineering Salmonella vaccine
 
Issue Date2011
 
PublisherSpringer. The Journal's web site is located at http://link.springer.de/link/service/journals/00253/index.htm
 
CitationApplied Microbiology And Biotechnology, 2011, v. 91 n. 1, p. 177-188 [How to Cite?]
DOI: http://dx.doi.org/10.1007/s00253-011-3317-0
 
AbstractLive attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains. © 2011 Springer-Verlag.
 
ISSN0175-7598
2012 Impact Factor: 3.689
2012 SCImago Journal Rankings: 1.307
 
DOIhttp://dx.doi.org/10.1007/s00253-011-3317-0
 
ISI Accession Number IDWOS:000291600600015
Funding AgencyGrant Number
RFCID
NSFC
Hong Kong Research Grants CouncilN_HKU 719/08
CRCG
Funding Information:

This work was supported by RFCID grants to JDH, a grant from NSFC and the Hong Kong Research Grants Council (N_HKU 719/08) to JDH and ES, and a grant from CRCG Seed Funding Programme for Applied Research to JDH.

 
ReferencesReferences in Scopus
 
GrantsToward the treatment of breast cancer by targeting breast tumor initiating cells with microRNAs
 
DC FieldValue
dc.contributor.authorYu, B
 
dc.contributor.authorYang, M
 
dc.contributor.authorWong, HYB
 
dc.contributor.authorWatt, RM
 
dc.contributor.authorSong, E
 
dc.contributor.authorZheng, BJ
 
dc.contributor.authorYuen, KY
 
dc.contributor.authorHuang, JD
 
dc.date.accessioned2011-09-23T05:43:07Z
 
dc.date.available2011-09-23T05:43:07Z
 
dc.date.issued2011
 
dc.description.abstractLive attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains. © 2011 Springer-Verlag.
 
dc.description.natureLink_to_subscribed_fulltext
 
dc.identifier.citationApplied Microbiology And Biotechnology, 2011, v. 91 n. 1, p. 177-188 [How to Cite?]
DOI: http://dx.doi.org/10.1007/s00253-011-3317-0
 
dc.identifier.citeulike9405864
 
dc.identifier.doihttp://dx.doi.org/10.1007/s00253-011-3317-0
 
dc.identifier.epage188
 
dc.identifier.hkuros192276
 
dc.identifier.isiWOS:000291600600015
Funding AgencyGrant Number
RFCID
NSFC
Hong Kong Research Grants CouncilN_HKU 719/08
CRCG
Funding Information:

This work was supported by RFCID grants to JDH, a grant from NSFC and the Hong Kong Research Grants Council (N_HKU 719/08) to JDH and ES, and a grant from CRCG Seed Funding Programme for Applied Research to JDH.

 
dc.identifier.issn0175-7598
2012 Impact Factor: 3.689
2012 SCImago Journal Rankings: 1.307
 
dc.identifier.issue1
 
dc.identifier.openurl
 
dc.identifier.pmid21611798
 
dc.identifier.scopuseid_2-s2.0-79959280769
 
dc.identifier.spage177
 
dc.identifier.urihttp://hdl.handle.net/10722/138958
 
dc.identifier.volume91
 
dc.languageeng
 
dc.publisherSpringer. The Journal's web site is located at http://link.springer.de/link/service/journals/00253/index.htm
 
dc.publisher.placeGermany
 
dc.relation.ispartofApplied Microbiology and Biotechnology
 
dc.relation.projectToward the treatment of breast cancer by targeting breast tumor initiating cells with microRNAs
 
dc.relation.referencesReferences in Scopus
 
dc.rightsThe original publication is available at www.springerlink.com
 
dc.subject.meshAntigens - genetics - metabolism
 
dc.subject.meshCloning, Molecular - methods
 
dc.subject.meshGene Expression
 
dc.subject.meshRecombination, Genetic
 
dc.subject.meshSalmonella typhi - genetics - metabolism
 
dc.subjectRecombineering Salmonella vaccine
 
dc.titleA method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy
 
dc.typeArticle
 
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Author Affiliations
  1. The University of Hong Kong
  2. Sun Yat-Sen University
  3. Prince Philip Dental Hospital