Molecular Biology Reports

, Volume 46, Issue 2, pp 1737–1746 | Cite as

Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos

  • Stefan WagnerEmail author
  • Judi McCracken
  • Sabine Bruszies
  • Ric Broadhurst
  • David N. Wells
  • Björn Oback
  • Jürgen Bode
  • Götz Laible
Original Article


Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircles for the generation of transgenic animals.


Scaffold/matrix attachment region Minicircle Episome Transgenic animals Plasmid 



We would like to thank Fleur Oback and Jan Oliver for assistance in SCNT, Jaime Oswald for outgrowing cells from blastocysts and Pauline Hunt for assembling and preparing the figures for this manuscript.


This work was supported by the Ministry of Business, Innovation and Employment (AgResearch Core Fund A16305, A13654 and A19064).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Foster K, Foster H, Dickson JG (2006) Gene therapy progress and prospects: Duchenne muscular dystrophy. Gene Ther 13(24):1677–1685CrossRefPubMedGoogle Scholar
  2. 2.
    Griesenbach U, Alton EW (2012) Progress in gene and cell therapy for cystic fibrosis lung disease. Curr Pharm Des 18(5):642–662CrossRefPubMedGoogle Scholar
  3. 3.
    Misaki W (2008) Bone marrow transplantation (BMT) and gene replacement therapy (GRT) in sickle cell anemia. Niger J Med 17(3):251–256CrossRefPubMedGoogle Scholar
  4. 4.
    Bank A (2008) On the road to gene therapy for beta-thalassemia and sickle cell anemia. Pediatr Hematol Oncol 25(1):1–4CrossRefPubMedGoogle Scholar
  5. 5.
    Gatehouse JA (2011) Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects. Curr Protein Pept Sci 12(5):409–416CrossRefPubMedGoogle Scholar
  6. 6.
    Muller M, Brem G (1998) Transgenic approaches to the increase of disease resistance in farm animals. Rev Sci Tech 17(1):365–378CrossRefPubMedGoogle Scholar
  7. 7.
    Anthony RV, Cantlon JD (2007) Ribonucleic acid interference: a new approach to the in vivo study of gene function. J Anim Sci 85(13 Suppl):E18–E19CrossRefPubMedGoogle Scholar
  8. 8.
    Mukai HY, Motohashi H, Ohneda O, Suzuki N, Nagano M, Yamamoto M (2006) Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation. Mol Cell Biol 26(21):7953–7965CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pravtcheva DD, Wise TL (1995) A postimplantation lethal mutation induced by transgene insertion on mouse chromosome 8. Genomics 30(3):529–544CrossRefPubMedGoogle Scholar
  10. 10.
    Magram J, Bishop JM (1991) Dominant male sterility in mice caused by insertion of a transgene. Proc Natl Acad Sci USA 88(22):10327–10331CrossRefPubMedGoogle Scholar
  11. 11.
    Kohn DB, Sadelain M, Glorioso JC (2003) Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer 3(7):477–488CrossRefPubMedGoogle Scholar
  12. 12.
    Dorer DR, Henikoff S (1994) Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell 77(7):993–1002CrossRefPubMedGoogle Scholar
  13. 13.
    Alonso-Gonzalez L, Couldrey C, Meinhardt MW, Cole SA, Wells DN, Laible G (2012) Primary transgenic bovine cells and their rejuvenated cloned equivalents show transgene-specific epigenetic differences. PLoS One 7(4):e35619CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Garrick D, Fiering S, Martin DI, Whitelaw E (1998) Repeat-induced gene silencing in mammals. Nat Genet 18(1):56–59CrossRefPubMedGoogle Scholar
  15. 15.
    Frederickson RM (2007) Integrating ideas on insertional mutagenesis by gene transfer vectors. Mol Ther 15(7):1228–1232CrossRefPubMedGoogle Scholar
  16. 16.
    Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res 27(2):426–428CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Bode J, Kohwi Y, Dickinson L, Joh T, Klehr D, Mielke C, Kohwi-Shigematsu T (1992) Biological significance of unwinding capability of nuclear matrix-associating DNAs. Science 255(5041):195–197CrossRefPubMedGoogle Scholar
  18. 18.
    Bode J, Winkelmann S, Gotze S, Spiker S, Tsutsui K, Bi C, Benham AKP C (2006) Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy. J Mol Biol 358(2):597–613CrossRefPubMedGoogle Scholar
  19. 19.
    Jenke BH, Fetzer CP, Stehle IM, Jonsson F, Fackelmayer FO, Conradt H, Bode J, Lipps HJ (2002) An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. EMBO Rep 3(4):349–354CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Argyros O, Wong SP, Harbottle RP (2011) Non-viral episomal modification of cells using S/MAR elements. Expert Opin Biol Ther 11(9):1177–1191CrossRefPubMedGoogle Scholar
  21. 21.
    Jenke AC, Scinteie MF, Stehle IM, Lipps HJ (2004) Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation. Mol Biol Rep 31(2):85–90CrossRefPubMedGoogle Scholar
  22. 22.
    Broll S, Oumard A, Hahn K, Schambach A, Bode J (2010) Minicircle performance depending on S/MAR-nuclear matrix interactions. J Mol Biol 395(5):950–965CrossRefPubMedGoogle Scholar
  23. 23.
    Nehlsen K, Broll S, Bode J (2006) Replicating minicircles: generation of nonviral episomes for the efficient modification of dividing cells. Gene Ther Mol Biol 10:233–244Google Scholar
  24. 24.
    Riu E, Chen ZY, Xu H, He CY, Kay MA (2007) Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo. Mol Ther 15(7):1348–1355CrossRefPubMedGoogle Scholar
  25. 25.
    Jenke AC, Stehle IM, Herrmann F, Eisenberger T, Baiker A, Bode J, Fackelmayer FO, Lipps HJ (2004) 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 101(31):11322–11327CrossRefPubMedGoogle Scholar
  26. 26.
    Stehle IM, Scinteie MF, Baiker A, Jenke AC, Lipps HJ (2003) Exploiting a minimal system to study the epigenetic control of DNA replication: the interplay between transcription and replication. Chromosome Res 11(5):413–421CrossRefPubMedGoogle Scholar
  27. 27.
    Buchholz F, Angrand PO, Stewart AF (1996) A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs. Nucleic Acids Res 24(15):3118–3119CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Brophy B, Smolenski G, Wheeler T, Wells D, L’Huillier P, Laible G (2003) Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein. Nat Biotechnol 21(2):157–162CrossRefPubMedGoogle Scholar
  29. 29.
    Oback B, Wiersema AT, Gaynor P, Laible G, Tucker FC, Oliver JE, Miller AL, Troskie HE, Wilson KL, Forsyth JT et al (2003) Cloned cattle derived from a novel zona-free embryo reconstruction system. Cloning Stem Cells 5(1):3–12CrossRefPubMedGoogle Scholar
  30. 30.
    Hogan B, Beddington R, Costantini F, Lacy E (1994) Manipulating the mouse embryo: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  31. 31.
    Argyros O, Wong SP, Niceta M, Waddington SN, Howe SJ, Coutelle C, Miller AD, Harbottle RP (2008) Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector. Gene Ther 15(24):1593–1605CrossRefPubMedGoogle Scholar
  32. 32.
    Haase R, Argyros O, Wong SP, Harbottle RP, Lipps HJ, Ogris M, Magnusson T, Vizoso Pinto MG, Haas J, Baiker A (2010) pEPito: a significantly improved non-viral episomal expression vector for mammalian cells. BMC Biotechnol 10:20CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Hagedorn C, Schnodt-Fuchs M, Boehme P, Abdelrazik H, Lipps HJ, Buning H (2017) S/MAR element facilitates episomal long-term persistence of adeno-associated virus vector genomes in proliferating cells. Hum Gene Ther 28(12):1169–1179CrossRefPubMedGoogle Scholar
  34. 34.
    Koirala A, Conley SM, Naash MI (2014) Episomal maintenance of S/MAR-containing non-viral vectors for RPE-based diseases. Adv Exp Med Biol 801:703–709CrossRefPubMedGoogle Scholar
  35. 35.
    Lin Y, Li Z, Wang T, Wang X, Wang L, Dong W, Jing C, Yang X (2015) MAR characteristic motifs mediate episomal vector in CHO cells. Gene 559(2):137–143CrossRefPubMedGoogle Scholar
  36. 36.
    Lufino MM, Manservigi R, Wade-Martins R (2007) An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency. Nucleic Acids Res 35(15):e98CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Ronald JA, Cusso L, Chuang HY, Yan X, Dragulescu-Andrasi A, Gambhir SS (2013) Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies. PLoS ONE 8(8):e73138CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Verghese SC, Goloviznina NA, Skinner AM, Lipps HJ, Kurre P (2014) S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection. Nucleic Acids Res 42(7):e53CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Wong SP, Argyros O, Harbottle RP (2015) Sustained expression from DNA vectors. Adv Genet 89:113–152CrossRefPubMedGoogle Scholar
  40. 40.
    Xu Z, Chen F, Zhang L, Lu J, Xu P, Liu G, Xie X, Mu W, Wang Y, Liu D (2016) Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells. Sci China Life Sci 59(10):1024–1033CrossRefPubMedGoogle Scholar
  41. 41.
    Grimes BR, Steiner CM, Merfeld-Clauss S, Traktuev DO, Smith D, Reese A, Breman AM, Thurston VC, Vance GH, Johnstone BH et al (2009) Interphase FISH demonstrates that human adipose stromal cells maintain a high level of genomic stability in long-term culture. Stem Cells Dev 18(5):717–724CrossRefPubMedGoogle Scholar
  42. 42.
    Olifent M, Miller A, Beaton A, L’Huillier P, Wells DN, Laible G (2002) Karyotyping as an important screen for suitable donor cells to generate cloned transgenic animals by nuclear transfer. Proc NZ Soc Anim Prod 62:199Google Scholar
  43. 43.
    Wells DN, Laible G, Tucker FC, Miller AL, Oliver JE, Xiang T, Forsyth JT, Berg MC, Cockrem K, L’Huillier PJ et al (2003) Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology 59(1):45–59CrossRefPubMedGoogle Scholar
  44. 44.
    Iqbal K, Barg-Kues B, Broll S, Bode J, Niemann H, Kues W (2009) Cytoplasmic injection of circular plasmids allows targeted expression in mammalian embryos. Biotechniques 47(5):959–968CrossRefPubMedGoogle Scholar
  45. 45.
    Manzini S, Vargiolu A, Stehle IM, Bacci ML, Cerrito MG, Giovannoni R, Zannoni A, Bianco MR, Forni M, Donini P et al (2006) Genetically modified pigs produced with a nonviral episomal vector. Proc Natl Acad Sci USA 103(47):17672–17677CrossRefPubMedGoogle Scholar
  46. 46.
    Eghbalsaied S, Ghaedi K, Laible G, Hosseini SM, Forouzanfar M, Hajian M, Oback F, Nasr-Esfahani MH, Oback B (2013) Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos. Reproduction 145(1):97–108CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.AgResearch Limited, Ruakura Research CentreHamiltonNew Zealand
  2. 2.Rowett InstituteUniversity of AberdeenAberdeenUK
  3. 3.Hannover Medical School (MHH)HannoverGermany

Personalised recommendations