Abstract
A tissue-specific transgenic model was employed to test the effects of intron and vector sequences on transgene expression in zebrafish after microinjection. In this model, the 2.3 kb promoter taken from the 5′ upstream region of the transcription initiation site of keratin 4 (krt4) was used to drive the enhanced green fluorescence protein (EGFP) reporter gene in a transgenic vector. For assaying the strength of EGFP expression, the effects of including an intron before the EGFP coding region or using different forms of DNA, including circular plasmid, linear full-length plasmid, and the linear transgene coding region without any prokaryotic vector sequence, were tested. After microinjection, the transgene expression was analyzed using transient assays. Consequently, further comparative analysis supported by Fisher’s exact test was performed based on the data generated by analyzing the strength of the transgene expression. It was shown that inclusion of an intron in the construct increases the transgene expression in a transient transgenic zebrafish assay. Furthermore, the circular plasmid containing the transgene produced the strongest EGFP expression.
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References
Amsterdam A, Lin S et al (1996) Requirements for green fluorescent protein detection in transgenic zebrafish embryos. Gene 173:99–103
Brinster RL, Chen HY et al (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci USA 82:4438–4442
Chen Y-H, Lee W-C et al (2001) Molecular structure, dynamic expression, and promoter analysis of zebrafish (Danio rerio) myf-5 gene. Genesis 29:22–35
Choi T, Huang M et al (1991) A generic intron increases gene expression in transgenic mice. Mol Cell Biol 11:3070–3074
Cretekos CJ, Grunwald DJ (1999) Alyron, an insertional mutation affecting early neural crest development in zebrafish. Dev Biol 210:322–338
Du SJ, Dienhart M (2001) Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos. Dev Dyn 222:655–666
Etchberger JF, Hobert O (2008) Vector-free DNA constructs improve transgene expression in C. elegans. Nat Methods 5:3
Gong Z, Ju B et al (2002) Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8. Dev Dyn 223:204–215
Grunwald DJ, Eisen JS (2002) Headwaters of the zebrafish—emergence of a new model vertebrate. Nat Rev Genet 3:717–724
Ju B, Xu Y et al (1999) Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters. Dev Genet 25:158–167
Kimmel CB, Ballard WW et al (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310
Kjer-Nielsen L, Holmberg K et al (1992) Impaired expression of chimaeric major histocompatibility complex transgenes associated with plasmid sequences. Transgenic Res 1:182–187
Ku N-O, Darling JM et al (2003) Keratin 8 and 18 mutations are risk factors for developing liver disease of multiple etiologies. Proc Natl Acad Sci USA 100:6063–6068
Niwa H, Miyazaki J-I et al (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372–376
Nott A, Meislin SH et al (2003) A quantitative analysis of intron effects on mammalian gene expression. RNA 9:607–617
Parinov S, Kondrichin I et al (2004) Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev Dyn 231:449–459
Rafiq M, Suen CKM et al (1997) Expression of recombinant human ceruloplasmin—an absolute requirement for splicing signals in the expression cassette. FEBS Lett 407:132–136
Sprague J, Doerry E et al (2001) The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research. Nucleic Acids Res 29:87–90
Sprague J, Clements D et al (2003) The Zebrafish Information Network (ZFIN): the zebrafish model organism database. Nucleic Acids Res 31:241–243
Stuart GW, McMurray JV et al (1988) Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103:403–412
Udvadia AJ, Linney E (2003) Windows into development: historic, current, and future perspectives on transgenic zebrafish. Dev Biol 256:1–17
Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish Danio (Brachydanio) Rerio. Univ. of Oregon Press, Eugene
Wixon J (2000) Featured organism: Danio rerio, the zebrafish. Yeast (Chichester, England) 17:225–231
Zeng Z, Liu X et al (2005) Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters. Dev Dyn 234:387–392
Acknowledgments
We are grateful to the Biomedical Research Council (BMRC), Agency for Science, Technology and Research (A*STAR) for funding. S.C. is supported by a NUS graduate research scholarship. We are grateful to Song Jie for technical support.
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Chatterjee, S., Min, L., Karuturi, R.K.M. et al. The role of post-transcriptional RNA processing and plasmid vector sequences on transient transgene expression in zebrafish. Transgenic Res 19, 299–304 (2010). https://doi.org/10.1007/s11248-009-9312-x
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DOI: https://doi.org/10.1007/s11248-009-9312-x