Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring β-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.
Abstract
To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring β-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.
참고문헌 (28)
- Baup D, "Variegation and silencing in a lentiviral-based murine transgenic model", Transgenic Res, 19, pp.399~414 (2010)
- Bishop JO, "Chromosomal insertion of foreign DNA", Repd Nutr Dev, 36, pp.607~618 (1996)
- Casco-Robles MM, "Simple and efficient transgenesis with I-SceI meganuclease in the newt, Cynops pyrrhogaster", Dev Dyn, 239, pp.3275~3284 (2010)
- Cho YS, "Characterization of estrogenresponsive transgenic marine medaka Oryzias dancena germlines harboring red fluorescent protein gene under the control by endogenous choriogenin H promoter", Transgenic Res, 22, pp.501~517 (2013)
- Cho YS, "Characterization of stable fluorescent transgenic marine medaka(Oryzias dancena)lines carrying red fluorescent protein gene driven by myosin light chain 2 promoter", Transgenic Res, 22, pp.849~859 (2013)
- Cho YS, "Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena", Transgenic Res, 20, pp.1333~1335 (2011)
- Dorer DR, "Transgene repeat arrays interact with distant heterochromatin and cause silencing in cis and trans", Genetics, 147, pp.1181~1190 (1997)
- Dunham RA, "Transgenic animal technology - A laboratory handbook", pp.305~334 (2014), Elsevier Inc
- Feng S, "Determining DNA methylation profiles using sequencing", Methods Mol Biol, 733, pp.223~238 (2011)
- Geurts AM, "Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers", PLoS Genet, 2, pp.e156~ (2006)
- Gibbs PD, "An in vivo screen for the luciferase transgene in zebrafish", Mol Mar Biol Biotechnol, 3, pp.307~316 (1994)
- Grabher C, "Meganuclease and transposon mediated transgenesis in medaka", Genome Biol, 8, pp.S10~ (2007)
- Hackett PB, "Recent advances in marine biotechnology, Vol 4", pp.77~145 (2000), Science Publishers
- Iyengar A, "Regulation and expression of transgenes in fish – a review", Transgenic Res, 5, pp.147~166 (1996)
- Kim DS, "Comparative analysis of inherited patterns of the transgene in transgenic mud loach Misgurnus mizolepis lines carrying the CAT reporter gene", Fish Sci, 70, pp.201~210 (2004)
- Koetsier PA, "Stability of transgene methylation patterns in mice : position effects, strain specificity and cellular mosaicism", Transgenic Res, 54, pp.235~244 (1996)
- Kong Q, "Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs", PLoS One, 4 (2009)
- 이상윤, "Molecular Characterization of Cytoskeletal Beta-Actin and its Promoter in the Javanese Ricefish Oryzias javanicus", Fisheries and Aquatic Sciences, 15(4), pp.317~324 (2012), 한국수산과학회
- Matzke MA, "Transgene silencing by the host genome defense : implications for the evolution of epigenetic control mechanisms in plants and vertebrates", Plant Mol Biol, 43, pp.401~415 (2000)
- Nam YK, "Environmental risk assessment for genetically modified organisms, Vol 3", pp.61~94 (2007), CABI Press
- Nam YK, "Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis", Transgenic Res, 10, pp.353~362 (2001)
- Nam, YK, "Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach(Misgurnus mizolepis)", Aquaculture, 172, pp.229~245 (1999)
- Pena RN, "Transgene methylation in mice reflects copy number but not expression level", Mol Biotechnol, 26, pp.215~219 (2004)
- Rembold M, "Transgenesis in fish : efficient selection of transgenic fish by co-injection with a fluorescent reporter construct", Nat Protoc, 1, pp.1133~1139 (2006)
- Schnetz K, "Cleavage by EcoO109 and DraII is inhibited by overlapping dcm methylation", Nucleic Acids Res, 16, pp.1623~ (1988)
- Uh M, "Transgene constructs in coho salmon(Oncorhynchus kisutch)are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA", Transgenic Res, 15, pp.711~727 (2006)
- Wu B, "Characterization of transgene integration pattern in F4 hGH-transgenic common carp(Cyprinus carpio L. )", Cell Res, 15, pp.447~454 (2005)
- Würtele H, "Illegitimate DNA integration in mammalian cells", Gene Therapy, 10, pp.1791~1799 (2003)