Article thumbnail

High-level transgene expression by homologous recombination-mediated gene transfer

By Mélanie Grandjean, Pierre-Alain Girod, David Calabrese, Kaja Kostyrko, Marianne Wicht, Florence Yerly, Christian Mazza, Jacques S. Beckmann, Danielle Martinet and Nicolas Mermod


Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression

Topics: Methods Online
Publisher: Oxford University Press
OAI identifier:
Provided by: PubMed Central

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.

Suggested articles


  1. (2007). A comparative study of S/MAR prediction tools.
  2. (2008). A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnology and Bioengineering,
  3. (1999). A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells.
  4. (2005). An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene.
  5. (1985). An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene.
  6. (2002). An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells.
  7. (1986). Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays.
  8. (2008). Anchoring the genome.
  9. (1989). Bent DNA is a structural feature of scaffold-attached regions in Drosophila melanogaster interphase nuclei.
  10. (1998). Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila.
  11. (1992). Biological significance of unwinding capability of nuclear matrix-associating DNAs.
  12. (2004). Boundary elements and nuclear organization.
  13. (2001). BRCA1 and BRCA2 and the genetics of breast and ovarian cancer.
  14. (2004). Breakpoint cluster regions of the AML-1 and ETO genes contain MAR elements and are preferentially associated with the nuclear matrix in proliferating HEL cells.
  15. (2007). Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid-mediated transgene expression.
  16. (1997). Chromatin domains and nuclear compartments: establishing sites of gene expression in eukaryotic nuclei.
  17. (2004). Chromatin loops are selectively anchored using scaffold/matrix-attachment regions.
  18. (1988). Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold.
  19. (1998). Conspiracy of silence among repeated transgenes.
  20. (2006). Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy.
  21. (1995). Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation.
  22. (1983). Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells.
  23. (2002). DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination.
  24. (1997). DNA-PK: at the cross-roads of biochemistry and genetics.
  25. (1996). Dosage-dependent modification of position-effect variegation in Drosophila.
  26. (1995). Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.
  27. (2006). Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells.
  28. (2002). Evaluation of sequence motifs found in scaffold/matrix-attached regions (S/MARs).
  29. (1998). Facilitation of chromatin dynamics by SARs.
  30. (2003). From DNA structure to gene expression: mediators of nuclear compartmentalization and dynamics.
  31. (2006). Genetic characterization of CHO production host DG44 and derivative recombinant cell lines.
  32. (2007). Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells.
  33. (2000). H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation.
  34. (1986). High frequency targeting of genes to specific sites in the mammalian genome.
  35. (2004). HIV-1 integration sites are flanked by potential MARs that alone can act as promoters.
  36. (2001). Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
  37. (1998). Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.
  38. (2004). Homologous recombination-mediated double-strand break repair.
  39. (2001). Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms.
  40. (1998). Homology-directed repair is a major double-strand break repair pathway in mammalian cells.
  41. (2000). Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector.
  42. (2004). Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster.
  43. (1988). Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.
  44. (1987). Improved methods for the isolation of individual and clustered mitotic chromosomes.
  45. (2004). Improved recombinant gene expression in CHO cells using matrix attachment regions.
  46. (2006). In-silico prediction and observations of nuclear matrix attachment.
  47. (2003). Interaction of EBV latent origin of replication with the nuclear matrix: identification of S/MAR sequences and protein components.
  48. (1999). Interrelationships of transcriptional machinery with nuclear architecture.
  49. (2001). Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.
  50. (2003). Maintenance of heterochromatin by RNA interference of tandem repeats.
  51. (2009). MAR elements regulate the probability of epigenetic switching between active and inactive gene expression.
  52. (2001). Matrix attachment region-dependent function of the immunoglobulin mu enhancer involves histone acetylation at a distance without changes in enhancer occupancy.
  53. (2005). Matrix attachment regions as targets for retroviral integration.
  54. (1997). Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.
  55. (2003). Molecular views of recombination proteins and their control.
  56. (1993). Nature of DNA sequences at the attachment regions of genes to the nuclear matrix.
  57. (1985). Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.
  58. (2000). NP/NMP4 transcription factors have distinct osteoblast nuclear matrix subdomains.
  59. (2004). Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome.
  60. (1984). Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold.
  61. (2000). Overview of vector design for mammalian gene expression.
  62. (1982). Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.
  63. (1995). Position-dependent variegation of globin transgene expression in mice.
  64. (2002). Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.
  65. (1995). Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.
  66. (1989). Protein: DNA interactions at chromosomal loop attachment sites.
  67. (2008). Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.
  68. (2001). Re-defining the chromatin loop domain.
  69. (2003). Recruitment of heterogeneous nuclear ribonucleoprotein A1 in vivo to the LMP/TAP region of the major histocompatibility complex.
  70. (2006). Repression of mutagenesis by Rad51D-mediated homologous recombination.
  71. (2002). SATB1 targets chromatin remodelling to regulate genes over long distances.
  72. (1995). Scaffold/matrix-attached regions: structural properties creating transcriptionally active loci.
  73. (2007). Statistical significance of quantitative PCR.
  74. (1999). Stopped at the border: boundaries and insulators.
  75. (1983). The double-strand-break repair model for recombination.
  76. (2003). The insulator binding protein CTCF associates with the nuclear matrix.
  77. (1998). Thinking about a nuclear matrix.
  78. (2003). Tissue-specific nuclear architecture and gene expression regulated by SATB1.
  79. (2000). Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements).
  80. (1981). Transforming DNA integrates into the host chromosome.
  81. (2000). Use of matrix attachment regions (MARs) to minimize transgene silencing.
  82. (2005). Use of the chicken lysozyme 50 matrix attachment region to generate high producer CHO cell lines.