Abstract
We established a transgenic zebrafish strain expressing chicken lysozyme gene under the control of the Japanese flounder keratin gene promoter, and investigated its resistance to a pathogenic bacterial infection. To generate the lysozyme transgenic construct, Japanese flounder keratin promoter was linked to both the hen egg white (HEW) lyoszyme gene and green fluorescence protein (GFP) gene used as a selection marker for the transgenic strains, in a recombinant plasmid. The recombinant plasmid was microinjected into fertilized zebrafish eggs. In F2 transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and gill from the embryonic stage to the adult stage. The expressions of HEW lysozyme and GFP mRNA were confirmed in the liver and skin by RT-PCR. Western blot analysis showed that both HEW lysozyme and GFP were present in protein extracts from the liver of transgenic zebrafish, but not in protein extracts from the muscle. The lytic activity of protein extracts from the liver (assessed by a lysoplate assay using Micrococcus lysodeikticus as a substrate) was 1.75 times higher in F2 transgenic zebrafish than in the wild type. In a challenge experiment, 65% of the F2 transgenic fish survived an infection of Flavobacterium columnare and 60% survived an infection of Edwardsiella tarda, whereas 100% of the control fish were killed by both pathogens. However, the survival rates of the transgenic fish were not significantly higher when higher concentrations of bacteria were used.
Similar content being viewed by others
References
A Brenot KY King B Janowiak O Griffith MG Caparon (2004) ArticleTitleContribution of glutathione peroxidase to the virulence of Streptococcus pyogenes Infect Immun 72 408–413 Occurrence Handle14688122 Occurrence Handle1:CAS:528:DC%2BD2cXpvFGk Occurrence Handle10.1128/IAI.72.1.408-413.2004
JM Davis H Clay JL Lewis N Ghori P Herbomel L Ramakrishnan (2002) ArticleTitleReal-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos Immunity 17 693–702 Occurrence Handle12479816 Occurrence Handle1:CAS:528:DC%2BD38XpvVWgsLw%3D Occurrence Handle10.1016/S1074-7613(02)00475-2
AE Ellis (2001) ArticleTitleInnate host defense mechanisms of fish against viruses and bacteria Dev Comp Immunol 25 827–839 Occurrence Handle11602198 Occurrence Handle1:CAS:528:DC%2BD3MXmsFWkt7g%3D Occurrence Handle10.1016/S0145-305X(01)00038-6
J Hawiger (2001) ArticleTitleInnate immunity and inflammation: a transcriptional paradigm Immunol Res 23 99–109 Occurrence Handle11444396 Occurrence Handle1:CAS:528:DC%2BD3MXltFWqsLc%3D Occurrence Handle10.1385/IR:23:2-3:099
J Hikima I Hirono T Aoki (2003) The lysozyme gene in fish N Shimizu T Aoki Hirono (Eds) Aquatic Genomics Springer-Verlag Tokyo, Japan 301–309
P Jölles J Jölles (1984) ArticleTitleWhat’s new in lysozyme research? Always a model system, today as yesterday Mol Cell Biochem 63 165–189 Occurrence Handle6387440
F Liu Z Wen (2002) ArticleTitleCloning and expression pattern of the lysozyme C gene in zebrafish Mech Dev 113 69–72 Occurrence Handle11900976 Occurrence Handle1:CAS:528:DC%2BD38XitFejuro%3D Occurrence Handle10.1016/S0925-4773(01)00658-X
S Minagawa J Hikima I T Hirono Aoki (2001) ArticleTitleExpression of Japanese flounder c-type lysozyme cDNA in insect cells Dev Comp Immunol 25 439–444 Occurrence Handle11356223 Occurrence Handle1:CAS:528:DC%2BD3MXjsFWgt78%3D Occurrence Handle10.1016/S0145-305X(01)00013-1
MN Neely JD Pfeifer M Caparon (2002) ArticleTitleStreptococcus-zebrafish model of bacterial pathogenesis Infect Immun 70 3904–3914 Occurrence Handle12065534 Occurrence Handle1:CAS:528:DC%2BD38XkvV2ksbs%3D Occurrence Handle10.1128/IAI.70.7.3904-3914.2002
O'Toole R, Von Hofsten J, Rosqvist R, Olsson PE and Wolf-Watz H (2004) Visualisation of zebrafish infection by GFP-labelled Vibrio anguillarum. Microb Pathog 37: 41–46.
MG Prouty NE Correa LP Barker P Jagadeeswaran KE Klose (2003) ArticleTitleZebrafish-Mycobacterium marinum model for mycobacterial pathogenesis FEMS Microbiol Lett 225 177–182 Occurrence Handle12951238 Occurrence Handle1:CAS:528:DC%2BD3sXmvVyju74%3D Occurrence Handle10.1016/S0378-1097(03)00446-4
PK Qasba S Kumar (1997) ArticleTitleMolecular divergence of lysozymes and alpha-lactalbumin Crit Rev Biochem Mol Biol 32 255–306 Occurrence Handle9307874 Occurrence Handle1:CAS:528:DyaK2sXmtlCqs7w%3D
Sambrook J and Russell DW (2000) Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, New York.
A Sarmasik G Warr TT Chen (2002) ArticleTitleProduction of transgenic medaka with increased resistance to bacterial pathogens Mar Biotechnol 4 310–322 Occurrence Handle14961264 Occurrence Handle1:CAS:528:DC%2BD38XlslKms7w%3D Occurrence Handle10.1007/s10126-002-0023-z
Udvadia AJ and Linney E (2003) Windows into development: historic, current, and future perspecives on transgenic zebrafish. Dev Biol 256: 1–17.
AM Sar Particlevan der RJ Musters FJ Eeden Particlevan BJ Appelmelk CM Vandenbroucke-Grauls W Bitter (2003) ArticleTitleZebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections Cell Microbiol 5 601–11 Occurrence Handle12925130 Occurrence Handle10.1046/j.1462-5822.2003.00303.x
Yazawa R, Hirono I and T Aoki (2005a) Characterization of promoter activities of four different Japanese flounder promoters in Transgenic Zebrafish. Mar Biotech. 7: 625–633.
Yazawa R, Hirono I, Ohira T and T Aoki (2005b) Induction of Japanese flounder TNF promoter activity by LPS in zebrafish embryo. Mar Biotech. 7: 231–235.
Yazawa R, Hirono I, Yamamoto E and Aoki T (2005c) Development of a gene transfer technique for Japanese flounder fertilized eggs by particle gun bombardment. Fish Sci. 71: 869–874.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yazawa, R., Hirono, I. & Aoki, T. Transgenic Zebrafish Expressing Chicken Lysozyme Show Resistance against Bacterial Diseases. Transgenic Res 15, 385–391 (2006). https://doi.org/10.1007/s11248-006-0009-0
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11248-006-0009-0