Mammalian Artificial Chromosome Formation in Human Cells After Lipofection of a PAC Precursor

  • Jose I. de las Heras
  • Leonardo D’Aiuto
  • Howard Cooke
Part of the Methods in Molecular Biology book series (MIMB, volume 240)


An artificial chromosome is a synthetic structure that carries three fundamental components for its long-term survival, replication, and segregation after cell division. These components are telomeres, one or more replication origins, and a centromere. The creation of such a molecule became feasible initially in the budding yeast Saccharomyces cerevisiae, where replication origins and centromeric sequences are well defined. In this organism, autonomously replicating sequences (ARS) were isolated by their ability to allow replication of plasmids carrying them. Plasmids containing an ARS element are capable of extrachromosomal replication in selective conditions but are lost if selection is removed from the culture because of unequal segregation. Introduction of a functional centromere to an ARS plasmid provides mitotic stability to the resulting yeast artificial chromosome (YAC) (reviewed by Newlon in ref. 1).


Replication Origin Microfuge Tube Yeast Artificial Chromosome Autonomously Replicate Sequence Comb Tooth 
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  1. 1.
    Newlon, C. S. (1988) Yeast chromosome replication and segregation. FEMS Microbiol. Rev. 52, 568–601.Google Scholar
  2. 2.
    Farr, C.J., Stevanovic, M., Thomson, E. J., Goodfellow, P. N., and Cooke, H. J. (1992) Telomere-associated chromosome fragmentation: application in genome manipulation and analysis. Nat. Genet. 2, 275–282.PubMedCrossRefGoogle Scholar
  3. 3.
    Auriche, C., Donini, P., and Ascenzioni F. (2001) Molecular and cytological analysis of a 5.5 Mb minichromosome. EMBO Rep. Feb. 2, 102–107.CrossRefGoogle Scholar
  4. 4.
    Heller, R., Brown, K. E., Burgtorf C., and Brown W. R. (1996) Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage. Proc. Natl. Acad. Sci. USA 93, 7125–7130.PubMedCrossRefGoogle Scholar
  5. 5.
    Shen, M. H., Mee, P. J., Nichols, J., Yang, J., Brook, F., Gardner, R. L., et al. (2000) A structural defined mini-chromosome vector for the mouse germline. Curr. Biol. 10, 31–34.PubMedCrossRefGoogle Scholar
  6. 6.
    Murphy, T. D. and Karpen G. H. (1995) Localization of centromere function in a Drosophila minichromosome. Cell 82, 599–609.PubMedCrossRefGoogle Scholar
  7. 7.
    Patnaik, P. K., Axelrod, N., Van der Ploeg, L. H., and Cross, G. A. (1996) Artificial linear mini-chromosomes for Trypanosoma brucei. Nucleic Acids Res. 24, 668–675.PubMedCrossRefGoogle Scholar
  8. 8.
    Harrington, J. J., Van Bokkelen, G., Mays, R.W., Gustashaw, K., and Willard, H. F. (1997) Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat. Genet. 15, 345–355.PubMedCrossRefGoogle Scholar
  9. 9.
    Ikeno M., Grimes, B., Okazaki, T., Nakano, M., Saitoh, K., Hoshino, H., et al. (1998) Construction of YAC-based mammalian artificial chromosomes. Nat. Biotechnol. 16, 431–439.PubMedCrossRefGoogle Scholar
  10. 10.
    Henning K.A., Novotny E. A., Compton S. T., Guan X. Y., Liu P. P., and Ashlock M. A. (1999) Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human α-satellite and single-copy DNA sequences. Proc. Natl. Acad. Sci. USA 96, 592–597.PubMedCrossRefGoogle Scholar
  11. 11.
    Ebersole T. A., Ross A., Clark E., McGill, N., Schindelhauer D., Cooke, H., et al. (2000) Mammalian artificial chromosome formation from circular alphoid input DNA does not require telomere repeats. Hum. Mol. Genet. 9, 1623–1631.PubMedCrossRefGoogle Scholar
  12. 12.
    Ten Hagen, K. G., Gilbert, D. M., Willard, H. F., and Cohen, S. N. (1990) Replication timing of DNA sequences asociated with human centromeres and telomeres. Mol. Cell. Biol. 10, 6348–6355.PubMedGoogle Scholar
  13. 13.
    Frengen, E., Zhao, B., Howe, S., Weichenhan D., Osoegawa, K., Gjernes, E., et al. (2000) Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells. Genomics 68, 118–126.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Jose I. de las Heras
    • 1
  • Leonardo D’Aiuto
    • 1
  • Howard Cooke
    • 1
  1. 1.Chromosome Biology Section, MRC Human Genetics UnitWestern General HospitalEdinburghUK

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