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Applied Microbiology and Biotechnology

, Volume 76, Issue 3, pp 651–657 | Cite as

Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions

  • Tianyun Wang
  • Lexun XueEmail author
  • Weihong Hou
  • Baosheng Yang
  • Yurong Chai
  • Xiang Ji
  • Yafeng Wang
Applied Genetics and Molecular Biotechnology

Abstract

Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated from Dunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects of MARs on expression of transgenes in the stably transformed cells of D. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven by D. salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells of D. salina by electroporation. Twenty stably transformed colonies of D. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies of D. salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression may not be through increasing the transgene copy numbers.

Keywords

Chloramphenicol acetyltransferase Dunaliella salina Matrix attachment region Transgene expression 

Notes

Acknowledgments

This work was supported by the grants from the National Natural Science Foundation of China (no. 30470030; 30270031) and Special Foundation for Training of Doctoral Students from Institutions of Higher Learning, Ministry of Education of P.R. China (no. 20050459007) and carried out at the Henan Key Laboratory of Molecular Medicine, P.R.C.

References

  1. Allen GC, Hall GE Jr, Childs LC, Weissinger AK, Spiker S, Thompson WF (1993) Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell 5:603–613CrossRefGoogle Scholar
  2. Allen GC, Hall GE Jr, Michalowski S, Newman W, Spiker S, Weissinger AK, Thompson WF (1996) High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899–913CrossRefGoogle Scholar
  3. Allen GC, Spiker S, Thompson WF (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol Biol 43:361–376CrossRefGoogle Scholar
  4. Ben-Amotz A, Shaish A, Avron M (1989) Mode of action of the massively accumulated beta-carotene of Dunaliella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol 91:1040–1043CrossRefGoogle Scholar
  5. Borovsky D (2003) Trypsin-modulating oostatic factor: a potential new larvicide for mosquito control. J Exp Biol 206(Pt 21):3869–3875CrossRefGoogle Scholar
  6. Butaye KM, Goderis IJ, Wouters PF, Pues JM, Delaure SL, Broekaert WF, Depicker A, Cammue BP, De Bolle MF (2004) Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J 39:440–449CrossRefGoogle Scholar
  7. Cheng ZQ, Targolli J, Wu R (2001) Tobacco matrix attachment region sequence increased transgene expression levels in rice plants. Mol Breed 7:317–327CrossRefGoogle Scholar
  8. Fukuda Y, Ohme M, Shinshi H (1991) Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells. Plant Mol Biol 16:1–10CrossRefGoogle Scholar
  9. Geng DG, Han Y, Wang YQ, Wang P, Zhang LM, Li WB, Sun YR (2004) Construction of a system for the stable expression of foreign genes in Dunaliella salina. Acta Bot Sin 46:342–346Google Scholar
  10. Girod PA, Zahn-Zabal M, Mermod N (2005) Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines. Biotechnol Bioeng 91:1–11CrossRefGoogle Scholar
  11. Goetze S, Baer A, Winkelmann S, Nehlsen K, Seibler J, Maass K, Bode J (2005) Performance of genomic bordering elements at predefined genomic loci. Mol Cell Biol 25:2260–2272CrossRefGoogle Scholar
  12. Jiang GZ, Lu YM, Niu XL, Xue LX (2005) The actin gene promoter-driven bar as a dominant selectable marker for nuclear transformation of Dunalialla salina. Acta Genet Sin 32:424–433Google Scholar
  13. Kim JM, Kim JS, Park DH, Kang HS, Yoon J, Baek K, Yoon Y (2004) Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol 107:95–105CrossRefGoogle Scholar
  14. Laemmli UK, Kas E, Poljak L, Adachi Y (1992) Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr Opin Genet Dev 2:275–285CrossRefGoogle Scholar
  15. Lu YM, Jiang GZ, Niu XL, Hou GQ, Zhang GX, Xue LX (2004) Cloning and functional analyses of promoters of two carbonic anhydrase genes from Dunaliella salina. Acta Genet Sin 31:1157–1166Google Scholar
  16. Mayfield SP, Franklin SE, Lerner RA (2003) Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA 100:438–442CrossRefGoogle Scholar
  17. Mlynarova L, Hricova A, Loonen A, Nap JP (2003) The presence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing. Plant Cell 15:2203–2217CrossRefGoogle Scholar
  18. Namciu SJ, Blochlinger KB, Fournier RE (1998) Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol Cell Biol 18:2382–2391Google Scholar
  19. Neznanov N, Kohwi-Shigematsu T, Oshima RG (1996) Contrasting effects of the SATB1 core nuclear matrix attachment region and flanking sequences of the keratin 18 gene in transgenic mice. Mol Biol Cell 7:541–552Google Scholar
  20. Oh SJ, Jeong JS, Kim EH, Yi NR, Yi SI, Jang IC, Kim YS, Suh SC, Nahm BH, Kim JK (2005) Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants. Plant Cell Rep 24:145–154CrossRefGoogle Scholar
  21. Peach C, Velten J (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17:49–60CrossRefGoogle Scholar
  22. Petersen K, Leah R, Knudsen S, Cameron-Mills V (2002) Matrix attachment regions (MARs) enhance transformation frequencies and reduce variance of transgene expression in barley. Plant Mol Biol 49:45–58CrossRefGoogle Scholar
  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  24. Stief A, Winter DM, Stratling WH, Sippel AE (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343–345CrossRefGoogle Scholar
  25. Vain P, Worland B, Kohli A, Snape JW, Cristoun P, Allen GC, Thompson WF (1999) Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J 18:233–242CrossRefGoogle Scholar
  26. Van Leeuwen W, Mlynarova L, Nap JP, van der Plas LH, van der Krol AR (2001) The effect of MAR elements on variation in spatial and temporal regulation of transgene expression. Plant Mol Biol 47:543–554CrossRefGoogle Scholar
  27. Walker TL, Purton S, Becker DK, Collet C (2005) Microalgae as bioreactors. Plant Cell Rep 24:629–641CrossRefGoogle Scholar
  28. Wang TY, Hou WH, Chai YR, Ji X, Wang JM, Xue LX (2005) Nuclear matrices and matrix attachment regions from Green alga: Dunaliella salina. Acta Genet Sin 32:1312–1318Google Scholar
  29. Xie H, Xu PR, Jia YL, Li J, Lu ZM, Xue LX (2007) Cloning and heterologous expression of nitrate reductase genes from Dunaliella salina. J Appl Phycol (in press). DOI 10.1007/s10811-007-9162-y
  30. Zhong J, Liu SJ, Ma SS, Yang W, Hu YL, Wu Q, Lin ZP (2004) Effect of matrix attachment regions on resveratrol production in tobacco with transgene of stilbene synthase from Parthenocissus henryana. Acta Bot Sin 46:948–954Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Tianyun Wang
    • 1
    • 2
  • Lexun Xue
    • 1
    Email author
  • Weihong Hou
    • 1
  • Baosheng Yang
    • 2
  • Yurong Chai
    • 1
  • Xiang Ji
    • 1
  • Yafeng Wang
    • 1
  1. 1.Laboratory for Cell Biology, The First Affiliated HospitalZhengzhou UniversityZhengzhouPeople’s Republic China
  2. 2.Department of Biochemistry and Molecular BiologyXinxiang Medical CollegeXinxiangPeople’s Republic China

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