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

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Grandjean, Mélanie, Girod, Pierre-Alain, Calabrese, David, Kostyrko, Kaja, Wicht, Marianne, Yerly, Florence, Mazza, Christian, Beckmann, Jacques S. +2 more·2011·Nucleic Acids Research
DOI 10.1093/nar/gkr436PMC PMC3159483
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Abstract

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.

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  • Inclusion of epigenetic elements can increase the number of clones that have integrated the transgene with a more defined copy number of transgene per cell, thus accelerating the selection process.

    “Inclusion of these various epigenetic elements not only can help to increase the expression level of biotherapeutics, but can also increase the number of clones that have integrated the transgene with a more defined copy number of transgene per cell, thus accelerating the selection process (Grandjean et al., 2011;Harraghy et al., 2015).”

    From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Grandjean et al., 2011

    The Results section, “MAR elements increase the copy number of genome-integrated transgenes” (page not available), reports increased integration and higher copy numbers in CHO cell pools and individual clones with MAR-containing plasmids. It does not establish that more clones have a more defined copy number per cell or that clone selection is accelerated. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Inclusion of epigenetic elements can help increase the expression level of biotherapeutics.

    “Inclusion of these various epigenetic elements not only can help to increase the expression level of biotherapeutics, but can also increase the number of clones that have integrated the transgene with a more defined copy number of transgene per cell, thus accelerating the selection process (Grandjean et al., 2011;Harraghy et al., 2015).”

    From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Grandjean et al., 2011

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Research works printed but not linked: 82

  1. [1]Kalos M Fournier RE Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain Mol. Cell. Biol. 1995 15 198 207 7799927 PMC231934
  2. [2]Recillas-Targa F Pikaart MJ Burgess-Beusse B Bell AC Litt MD West AG Gaszner M Felsenfeld G Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities Proc Natl Acad Sci USA 2002 99 6883 6888 12011446 PMC124498
  3. [3]Robertson G Garrick D Wu W Kearns M Martin D Whitelaw E Position-dependent variegation of globin transgene expression in mice Proc. Natl Acad. Sci. USA 1995 92 5371 5375 7777514 PMC41696
  4. [4]Henikoff S Dosage-dependent modification of position-effect variegation in Drosophila Bioessays 1996 18 401 409 8639163
  5. [5]Wakimoto BT Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila Cell 1998 93 321 324 9590165
  6. [6]Martienssen RA Maintenance of heterochromatin by RNA interference of tandem repeats Nat. Genet. 2003 35 213 214 14593407
  7. [7]Bell AC Felsenfeld G Stopped at the border: boundaries and insulators Curr. Opin. Genet. Dev. 1999 9 191 198 10322134
  8. [8]Hart CM Laemmli UK Facilitation of chromatin dynamics by SARs Curr. Opin. Genet. Dev. 1998 8 519 525 9794825
  9. [9]Bode J Goetze S Heng H Krawetz SA Benham C From DNA structure to gene expression: mediators of nuclear compartmentalization and dynamics Chromosome Res. 2003 11 435 445 12971720
  10. [10]Bode J Benham C Knopp A Mielke C Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements) Crit. Rev. Eukaryot. Gene Expr. 2000 10 73 90 10813396
  11. [11]Gasser SM Laemmli UK Improved methods for the isolation of individual and clustered mitotic chromosomes Exp. Cell Res. 1987 173 85 98 3678386
  12. [12]Bode J Schlake T Rios-Ramirez M Mielke C Stengert M Kay V Klehr-Wirth D Scaffold/matrix-attached regions: structural properties creating transcriptionally active loci Int. Rev. Cytol. 1995 162A 389 454 8575884
  13. [13]Jackson DA Chromatin domains and nuclear compartments: establishing sites of gene expression in eukaryotic nuclei Mol. Biol. Rep. 1997 24 209 220 9291094
  14. [14]Stein GS van Wijnen AJ Stein JL Lian JB Interrelationships of transcriptional machinery with nuclear architecture Crit. Rev. Eukaryot. Gene Expr. 1999 9 183 190 10651235
  15. [15]Heng HH Krawetz SA Lu W Bremer S Liu G Ye CJ Re-defining the chromatin loop domain Cytogenet. Cell Genet. 2001 93 155 161 11528105
  16. [16]Heng HH Goetze S Ye CJ Liu G Stevens JB Bremer SW Wykes SM Bode J Krawetz SA Chromatin loops are selectively anchored using scaffold/matrix-attachment regions J. Cell Sci. 2004 117 999 1008 14996931
  17. [17]Mirkovitch J Mirault ME Laemmli UK Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold Cell 1984 39 223 232 6091913
  18. [18]Capelson M Corces VG Boundary elements and nuclear organization Biol. Cell 2004 96 617 629 15519696
  19. [19]Boulikas T Nature of DNA sequences at the attachment regions of genes to the nuclear matrix J. Cell Biochem. 1993 52 14 22 8320271
  20. [20]Homberger HP Bent DNA is a structural feature of scaffold-attached regions in Drosophila melanogaster interphase nuclei Chromosoma 1989 98 99 104 2505984
  21. [21]Girod PA Nguyen DQ Calabrese D Puttini S Grandjean M Martinet D Regamey A Saugy D Beckmann JS Bucher P Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells Nat. Methods 2007 4 747 753 17676049
  22. [22]Platts AE Quayle AK Krawetz SA In-silico prediction and observations of nuclear matrix attachment Cell Mol. Biol. Lett. 2006 11 191 213 16847565 PMC6276010
  23. [23]Evans K Ott S Hansen A Koentges G Wernisch L A comparative study of S/MAR prediction tools BMC Bioinformatics 2007 8 71 17335576 PMC1847452
  24. [24]Ottaviani D Lever E Takousis P Sheer D Anchoring the genome Genome Biol. 2008 9 201 18226181 PMC2395230
  25. [25]Koina E Piper A An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene J. Cell Biochem. 2005 95 391 402 15779006
  26. [26]Mearini G Chichiarelli S Zampieri M Masciarelli S D'Erme M Ferraro A Mattia E Interaction of EBV latent origin of replication with the nuclear matrix: identification of S/MAR sequences and protein components FEBS Lett. 2003 547 119 124 12860398
  27. [27]Amati BB Gasser SM Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold Cell 1988 54 967 978 2843296
  28. [28]Piechaczek C Fetzer C Baiker A Bode J Lipps HJ A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells Nucleic Acids Res. 1999 27 426 428 9862961 PMC148196
  29. [29]Jenke AC Stehle IM Herrmann F Eisenberger T Baiker A Bode J Fackelmayer FO Lipps HJ Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome Proc. Natl Acad. Sci. USA 2004 101 11322 11327 15272077 PMC509201
  30. [30]Liebich I Bode J Reuter I Wingender E Evaluation of sequence motifs found in scaffold/matrix-attached regions (S/MARs) Nucleic Acids Res. 2002 30 3433 3442 12140328 PMC137072
  31. [31]Pederson T Thinking about a nuclear matrix J. Mol. Biol. 1998 277 147 159 9514762
  32. [32]Cai S Han HJ Kohwi-Shigematsu T Tissue-specific nuclear architecture and gene expression regulated by SATB1 Nat. Genet. 2003 34 42 51 12692553
  33. [33]Feister HA Torrungruang K Thunyakitpisal P Parker GE Rhodes SJ Bidwell JP NP/NMP4 transcription factors have distinct osteoblast nuclear matrix subdomains J. Cell Biochem. 2000 79 506 517 10972987
  34. [34]Dunn KL Zhao H Davie JR The insulator binding protein CTCF associates with the nuclear matrix Exp. Cell Res. 2003 288 218 223 12878173
  35. [35]Fernandez LA Winkler M Grosschedl R Matrix attachment region-dependent function of the immunoglobulin mu enhancer involves histone acetylation at a distance without changes in enhancer occupancy Mol. Cell. Biol. 2001 21 196 208 11113195 PMC88794
  36. [36]Yasui D Miyano M Cai S Varga-Weisz P Kohwi-Shigematsu T SATB1 targets chromatin remodelling to regulate genes over long distances Nature 2002 419 641 645 12374985
  37. [37]Donev R Horton R Beck S Doneva T Vatcheva R Bowen WR Sheer D Recruitment of heterogeneous nuclear ribonucleoprotein A1 in vivo to the LMP/TAP region of the major histocompatibility complex J. Biol. Chem. 2003 278 5214 5226 12435746
  38. [38]Rajaiya J Nixon JC Ayers N Desgranges ZP Roy AL Webb CF Induction of immunoglobulin heavy-chain transcription through the transcription factor Bright requires TFII-I Mol. Cell. Biol. 2006 26 4758 4768 16738337 PMC1489113
  39. [39]Galbete JL Buceta M Mermod N MAR elements regulate the probability of epigenetic switching between active and inactive gene expression Mol. Biosyst. 2009 5 143 150 19156259
  40. [40]Kwaks TH Otte AP Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells Trends Biotechnol. 2006 24 137 142 16460822
  41. [41]Allen GC Spiker S Thompson WF Use of matrix attachment regions (MARs) to minimize transgene silencing Plant Mol. Biol. 2000 43 361 376 10999416
  42. [42]Namciu SJ Fournier RE Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster Mol. Cell. Biol. 2004 24 10236 10245 15542833 PMC529032
  43. [43]Dang Q Auten J Plavec I Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector J. Virol. 2000 74 2671 2678 10684282 PMC111756
  44. [45]Iarovaia OV Shkumatov P Razin SV 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 J. Cell Sci. 2004 117 4583 4590 15331666
  45. [46]Kulkarni A Pavithra L Rampalli S Mogare D Babu K Shiekh G Ghosh S Chattopadhyay S HIV-1 integration sites are flanked by potential MARs that alone can act as promoters Biochem. Biophys. Res. Commun. 2004 322 672 677 15325282
  46. [47]Johnson CN Levy LS Matrix attachment regions as targets for retroviral integration Virol. J. 2005 2 68 16111492 PMC1198263
  47. [48]Blasquez VC Sperry AO Cockerill PN Garrard WT Protein: DNA interactions at chromosomal loop attachment sites Genome 1989 31 503 509 2561108
  48. [49]Bae YS Kawasaki I Ikeda H Liu LF Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro Proc. Natl Acad. Sci. USA 1988 85 2076 2080 2832845 PMC279931
  49. [50]Keeney S Giroux CN Kleckner N Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family Cell 1997 88 375 384 9039264
  50. [51]Girod PA Zahn-Zabal M Mermod N Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines Biotechnol. Bioeng. 2005 91 1 11 15889435
  51. [52]Urlaub G Kas E Carothers AM Chasin LA Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells Cell 1983 33 405 412 6305508
  52. [53]Blunt T Finnie NJ Taccioli GE Smith GC Demengeot J Gottlieb TM Mizuta R Varghese AJ Alt FW Jeggo PA Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation Cell 1995 80 813 823 7889575
  53. [54]Hinz JM Tebbs RS Wilson PF Nham PB Salazar EP Nagasawa H Urbin SS Bedford JS Thompson LH Repression of mutagenesis by Rad51D-mediated homologous recombination Nucleic Acids Res. 2006 34 1358 1368 16522646 PMC1390685
  54. [55]Derouazi M Martinet D Besuchet Schmutz N Flaction R Wicht M Bertschinger M Hacker DL Beckmann JS Wurm FM Genetic characterization of CHO production host DG44 and derivative recombinant cell lines Biochem. Biophys. Res. Commun. 2006 340 1069 1077 16403443
  55. [56]Milligan L Antoine E Bisbal C Weber M Brunel C Forne T Cathala G H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation Oncogene 2000 19 5810 5816 11126368
  56. [57]Karlen Y McNair A Perseguers S Mazza C Mermod N Statistical significance of quantitative PCR BMC Bioinformatics 2007 8 131 17445280 PMC1868764
  57. [58]Akita H Ito R Kamiya H Kogure K Harashima H Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid-mediated transgene expression J. Gene Med. 2007 9 197 207 17351985
  58. [59]Kim JM Kim JS Park DH Kang HS Yoon J Baek K Yoon Y Improved recombinant gene expression in CHO cells using matrix attachment regions J. Biotechnol. 2004 107 95 105 14711493
  59. [60]Folger KR Thomas K Capecchi MR Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei Mol. Cell. Biol. 1985 5 59 69 2984556 PMC366678
  60. [62]Wyman C Ristic D Kanaar K Homologous recombination-mediated double-strand break repair DNA Repair 2004 3 827 833 15279767
  61. [63]Wong EA Capecchi MR Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays Somat. Cell Mol. Genet. 1986 12 63 72 3003931
  62. [64]Takata M Sasaki MS Sonoda E Morrison C Hashimoto M Utsumi H Yamaguchi-Iwai Y Shinohara A Takeda S 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 EMBO J. 1998 17 5497 5508 9736627 PMC1170875
  63. [65]Delacote F Han M Stamato TD Jasin M Lopez BS An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells Nucleic Acids Res. 2002 30 3454 3463 12140331 PMC137076
  64. [66]Clikeman JA Khalsa GJ Barton SL Nickoloff JA Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms Genetics 2001 157 579 589 11156980 PMC1461527
  65. [67]Allen C Kurimasa A Brenneman MA Chen DJ Nickoloff JA DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination Proc. Natl Acad. Sci. USA 2002 99 3758 3763 11904432 PMC122597
  66. [68]Pierce AJ Hu P Han M Ellis N Jasin M Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells Genes Dev. 2001 15 3237 3242 11751629 PMC312854
  67. [69]Jeggo PA DNA-PK: at the cross-roads of biochemistry and genetics Mutat Res. 1997 384 1 14 9201268
  68. [70]Kaufman RJ Overview of vector design for mammalian gene expression Mol. Biotechnol. 2000 16 151 160 11131974
  69. [71]Chusainow J Yang YS Yeo JH Toh PC Asvadi P Wong NS Yap MG A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnology and Bioengineering 2008 102 1182 1196 18979540
  70. [72]Henikoff S Conspiracy of silence among repeated transgenes Bioessays 1998 20 532 535 9723001
  71. [73]West SC Molecular views of recombination proteins and their control Nat. Rev. Mol. Cell Biol. 2003 4 435 445 12778123
  72. [74]Robins DM Ripley S Henderson AS Axel R Transforming DNA integrates into the host chromosome Cell 1981 23 29 39 7214526
  73. [75]Folger KR Wong EA Wahl G Capecchi MR Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules Mol. Cell Biol. 1982 2 1372 1387 6298598 PMC369942
  74. [76]Thomas KR Folger KR Capecchi MR High frequency targeting of genes to specific sites in the mammalian genome Cell 1986 44 419 428 3002636
  75. [77]Chen HH Ho YP Jiang X Mao HQ Wang TH Leong KW Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET Mol. Ther. 2008 16 324 332 18180773 PMC3014860
  76. [78]Jacquier A Dujon B An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene Cell 1985 41 383 394 3886163
  77. [79]Kostriken R Strathern JN Klar AJ Hicks JB Heffron F A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae Cell 1983 35 167 174 6313222
  78. [80]Szostak JW Orr-Weaver TL Rothstein RJ Stahl FW The double-strand-break repair model for recombination Cell 1983 33 25 35 6380756
  79. [81]Smih F Rouet P Romanienko PJ Jasin M Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells Nucleic Acids Res. 1995 23 5012 5019 8559659 PMC307507
  80. [82]Chevalier BS Stoddard BL Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility Nucleic Acids Res. 2001 29 3757 3774 11557808 PMC55915
  81. [83]Bode J Kohwi Y Dickinson L Joh T Klehr D Mielke C Kohwi-Shigematsu T Biological significance of unwinding capability of nuclear matrix-associating DNAs Science 1992 255 195 197 1553545
  82. [84]Bode J Winkelmann S Gotze S Spiker S Tsutsui K Bi C Prashanth AK Benham C Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy J. Mol. Biol. 2006 358 597 613 16516920

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Authors
Grandjean, M., Girod, P.-A., Calabrese, D., Kostyrko, K., Wicht, M., Yerly, F., Mazza, C., Beckmann, J. S., Martinet, D., & Mermod, N.
Affiliations
  • École Polytechnique Fédérale de Lausanne (CH)
  • University of Fribourg (CH)
Corresponding author
Mermod, Nicolas
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2011-06-06
Volume
39
Issue
15
Pages
e104-e104
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkr436
PMC
PMC3159483
OpenAlex
W2018218652
Cited by
53
References
2
Citations by year
2026: 1 · 2024: 2 · 2023: 4 · 2022: 4 · 2020: 3 · 2019: 2 · 2018: 4 · 2017: 2 · 2016: 5 · 2015: 9 · 2014: 5 · 2013: 8 · 2012: 2
FWCI
3.39
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • RERO DOC (Universität Bern) (submittedVersion)
  • reroDoc Digital Library (submittedVersion)
  • IRIS (submittedVersion, other-oa)
  • SERVAL (Université de Lausanne) (submittedVersion, other-oa)
Funders
  • Kommission für Technologie und Innovation
  • Université de Lausanne
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
homologous recombination, matrix attachment regions, transgene expression, epigenetic silencing, chromosomal integration, non-homologous end joining, cell cycle, tandem gene copies, plasmid vectors
MeSH terms
Animals, Cell Cycle, Cell Line, Cell Nucleus, DNA, Genetic Vectors, Plasmids, Recombination, Genetic, Sequence Homology, Nucleic Acid, Transfection, Gene Expression, Gene Dosage, Transgenes, Active Transport, Cell Nucleus, Matrix Attachment Regions

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