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Position‐independent expression of transgenes in zebrafish

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Abstract

The variability in expression patterns of transgenes, caused by the influence of neighboring chromatin, is called 'position effect'. Border elements are DNA sequences, which have the ability to alleviate position effects. The abilities of two types of border elements, scs/scs′ from the D. melanogaster 87A7 heat shock locus and the A‐element from the chicken lysozyme gene, to protect transgenes from position effects were quantified in developing zebrafish embryos. The transgenic construct used was FV3CAT, which consists of the carp β‐actin transcriptional regulatory region, the chloramphenicol acetyltransferase (CAT) gene and the 3′‐untranslated region from the Chinook salmon growth hormone gene. FV3CAT constructs flanked by either scs/scs′‐elements or A‐elements were introduced into zebrafish chromosomes and the spatial and temporal expression patterns of the transgenes were quantified in multiple generations of transgenic zebrafish. Levels of transgene expression were uniform in the pre‐differentiated and fully differentiated populations of cells present during embryonic development. Levels of transgene expression were proportional to the numbers of integrated transgenes. Expression of transgenes per cell varied less than two‐fold in different transgenic lines. Both types of border elements were able to prevent the influences of neighboring chromatin on transgene expression through three generations of fish. The results are consistent with the ability of border elements to function with equal efficiencies in the many cell types found in vertebrates. Thus, inclusion of border elements in genetic constructs can provide reliable and reproducible levels of gene expression in multiple lines of fish.

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References

  • Amsterdam A, Lin S and Hopkins N (1995) The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev Biol 171: 123–129.

    Google Scholar 

  • Amsterdam A, Lin S, Moss LG and Hopkins N (1996) Requirements for green fluorescent protein detection in transgenic zebrafish embryos. Gene 173: 99–103.

    Google Scholar 

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K (1987) Current Protocols in Molecular Biology. Wiley, New York.

    Google Scholar 

  • Bayer TA and Campos-Ortega JA (1992) A transgene containing lacZ is expressed in primary sensory neurons in zebrafish. Development 115: 421–426.

    Google Scholar 

  • Bonifer C, Huber MC, Faust N and Sippel AE (1996a) Regulation of the chicken lysozyme locus in transgenic mice. Crit Rev Eukaryot Gene Expr 6: 285–297.

    Google Scholar 

  • Bonifer C, Huber MC, Jagle U, Faust N and Sippel AE (1996b) Prerequisites for tissue specific and position independent expression of a gene locus in transgenic mice. J MolMed 74: 663–671.

    Google Scholar 

  • Bonifer C, Yannoutsos N, Kruger G, Grosveld F and Sippel AE (1994) Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice. Nucleic Acids Res 22: 4202–4210.

    Google Scholar 

  • Cai H and Levine M (1995) Modulation of enhancer–promoter interactions by insulators in the Drosophila embryo. Nature 376: 533–536.

    Google Scholar 

  • Caldovic L (1998) Position-independent expression and germlinetransmission of transgenes in zebrafish. PhD thesis, University of Minnesota, St. Paul, USA.

    Google Scholar 

  • Caldovic L and Hackett PB (1995) Development of positionindependent expression vectors and their transfer into transgenic fish. Mol Mar Biol Biotechnol 4: 51–61.

    Google Scholar 

  • Chung JH, Bell AC and Felsenfeld G (1997) Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci USA 94: 575–580.

    Google Scholar 

  • Chung JH, Whiteley M and Felsenfeld G (1993) A 50 element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74: 505–514.

    Google Scholar 

  • Culp P, Nusslein-Volhard C and Hopkins N (1991) High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs. Proc Natl Acad Sci USA 88: 7953–7957.

    Google Scholar 

  • Dobie K, Mehtali M, McMlenaghan M and Lathe R (1997) Variegated gene expression in mice. Trends Genet 13: 127–130.

    Google Scholar 

  • Eisen JS (1996) Zebrafish make a big splash. Cell 87: 969–977.

    Google Scholar 

  • Fahrenkrug SC, Dahlquist MO, Clark KJ, Joshi B, Jagus R and Hackett PB (1999) Embryonic expression and genomic organization of zebrafish translation initiation factor 4E. Differentiation (submitted).

  • Geyer PK (1997) The role of insulator elements in defining domains of gene expression. Curr Op Genet Devel 7: 242–248.

    Google Scholar 

  • Gibbs PD, Gray A and Thorgaard G (1994a) Inheritance of p element and reporter gene sequences in zebrafish. Mol Mar Biol Biotechnol 3: 317–326.

    Google Scholar 

  • Gibbs PD, Peek A and Thorgaard G (1994b) An in vivo screen for the luciferase transgene in zebrafish. Mol Mar Biol Biotechnol 3: 307–316.

    Google Scholar 

  • Hackett PB (1993) The molecular biology of transgenic fish. In: Hochachka P and Mommsen T (eds) Biochemistry and Molecular Biology of Fishes, Vol. 2, pp. 207–240.

  • Hackett PB, Iszvak Z, Ivics Z and Caldovic L (1999) Development of genetic tools for transgenic animals. In: Murray JD, Anderson GB, McGloughlin MM and Oberharuer AM (eds) Transgenic Animals in Agriculture, pp. 19–35, CAB International, Wallingford.

    Google Scholar 

  • Hackett PB and Alvarez MC (1999) The molecular genetics of transgenic fish. Adv Mar Biotech 4: (in press).

  • Henikoff S (1998) Conspiracy of silence among repeated transgenes. BioEssays 20: 532–534.

    Google Scholar 

  • Higashijima S, Okamoto H, Ueno N, Hotta Y and Eguchi G (1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol 192: 289–299.

    Google Scholar 

  • Kellum R and Schedl P (1991) A position-effect assay for boundaries of higher order chromosomal domains. Cell 64: 941–950.

    Google Scholar 

  • Kimmel CB (1989) Genetics and early development of zebrafish. Trends Gen 5: 283–288.

    Google Scholar 

  • Krebs JE and Dunaway M (1998) The scs and scs0 insulator elements impart a cis requirement on enhancer-promoter interactions. Mol Cell 1: 301–308.

    Google Scholar 

  • Levy-Wilson B (1995) Transcriptional control of the human apolipoprotein B gene in cell culture and in transgenic animals. Prog Nucleic Acis Res Mol Biol 50: 161–190.

    Google Scholar 

  • Lin S, Gaiano N, Culp P, Burns JC, Friedmann T, Yee JK and Hopkins N (1994a) Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265: 666–669.

    Google Scholar 

  • Lin S, Yang S and Hopkins N (1994b) lacZ expression in germline transgenic zebrafish can be detected in living embryos. Dev Biol 161: 77–83.

    Google Scholar 

  • Liu ZJ, Moav B, Faras AJ, Guise KS, Kapuscinski AR and Hackett PB (1990a) Development of expression vectors for transgenic fish. Biotechnology (NY) 8: 1268–1272.

    Google Scholar 

  • Liu ZJ, Moav B, Faras AJ, Guise KS, Kapuscinski AR and Hackett PB (1990b) Functional analysis of elements affecting expression of the beta-actin gene of carp. Mol Cell Biol 10: 3432–3440.

    Google Scholar 

  • Long Q, Meng A, Wang H, Jessen JR, Farrel MJ and Lin S (1997) GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124: 4105–4111.

    Google Scholar 

  • Maclean N (1998) Regulation and exploitation of transgenes in fish. Mut Res 399: 255–266.

    Google Scholar 

  • McKnight RA, Shamay A, Sankaran L, Wall RJ and Hennighausen L (1992) Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci USA 89, 6943–6947.

    Google Scholar 

  • McKnight RA, Spencer M, Wall RJ and Hennighausen L (1996) Severe position effects imposed on a 1 kb mouse whey acidic protein gene promoter are overcome by heterologous matrix attachment regions. Mol Reprod Dev 44, 179–184.

    Google Scholar 

  • Miynarova L, Loonen A, Heldens J, Jansen RC, Keizer P, Stiekema WJ and Nap JP (1994) Reduced position effect in mature transgenic plants conferred by the chicken lysozyme matrixassociated region. Plant Cell 6, 417–426.

    Google Scholar 

  • Morris JR, Chen J-L, Geyer PK and Wu C-T (1998) Two modes of transvection: Enhancer action in trans and bypass of a chromatin insulator in cis. Proc Natl Acad Sci USA 95: 10740–10745.

    Google Scholar 

  • Namciu SJ, Blochlinger KB and Fournier REK (1998) Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol Cell Biol 18: 2382–2391.

    Google Scholar 

  • Phi-Van L, von Kries JP, Ostertag W and Stratling WH (1990) The chicken lysozyme 50 matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol 10: 2302–2307.

    Google Scholar 

  • Pikaart MJ, Recillas-Targa F and Felsenfeld G (1998) Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 12: 2852–2862.

    Google Scholar 

  • Poljak L, Seum C, Mattioni T and Laemmli UK (1994) SARs stimulate but do not confer position independent gene expression. Nucleic Acids Res 22: 4386–4394.

    Google Scholar 

  • Roseman RR, Pirrotta V and Geyer PK (1993) The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J 12: 435–442.

    Google Scholar 

  • Scott KS and Geyer PK (1995) Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes. EMBO J 14: 6258–6267.

    Google Scholar 

  • Stief A, Winter DM, Stratling WH and Sippel AE (1989) A nuclear DNA attachment element mediates elevated and positionindependent gene activity. Nature 341, 343–345.

    Google Scholar 

  • Stuart GW, McMurray JV and Westerfield M (1988) Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103: 403–412.

    Google Scholar 

  • Stuart GW, Vielkind JR, McMurray JV and Westerfield M (1990) Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development 109: 577–584.

    Google Scholar 

  • Thompson EM, Christians E, Stinnakre MG and Renard JP (1994) Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues. Mol Cell Biol 14: 4694–4703.

    Google Scholar 

  • Westerfield M (1995) The Zebrafish Book. University of Oregon Press, Eugene, USA.

    Google Scholar 

Download references

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Authors and Affiliations

  1. Department of Genetics Cell Biology and Development, University of Minnesota, St. Paul, MN, 55108‐1095

    Ljubica Caldovic, Dritan Agalliu & Perry B. Hackett

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  1. Ljubica Caldovic
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  2. Dritan Agalliu
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  3. Perry B. Hackett
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Caldovic, L., Agalliu, D. & Hackett, P.B. Position‐independent expression of transgenes in zebrafish. Transgenic Res 8, 321–334 (1999). https://doi.org/10.1023/A:1008993022331

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