Artificial Chromosomes


Artificial chromosomes are DNA molecules assembled in vitro from defined constituents, which guarantee stable maintenance of large DNA fragments with the properties of natural chromosomes. Artificial chromosomes are useful for genome sequencing programmes, for functional characterization of entire genomic regions and for the transduction of large DNA segments into human and nonhuman mammalian cells.

Keywords: BAC; YAC; PAC; MAC; Epstein‐Barr virus

Figure 1.

Individual components of the widely used yeast artificial chromosome (YAC) vector pYAC4, bacterial artificial chromosome (BAC) vector pBeloBAC11, and P1 phage‐derived artificial chromosome (PAC) vector pCYPAC‐1. CEN, centromeres; TEL, telomeres; ARS, autonomously replicating sequence (yeast ‘origin of replication’); AmpR (KanR, ChlR), ampicillin (kanamycin, chloramphenicol) resistance gene; TRP1 and URA3, yeast selectable marker genes; parA, parB, parC, repE, F plasmid regulatory genes; oriS, F plasmid origin of replication; cosN, phage λ terminase restriction site; loxP, loxP recombination site; lacZ, β galactosidase reporter gene; T7 and SP6, sequencing primer regions.

Figure 2.

Structural map of ‘top‐down’ engineered mammalian artificial chromosome (MAC) systems. Left: dissection of mammalian chromosomes into mini‐chromosomes by targeted chromosome fragmentation (TCF). Either alphoid centromeric repeats (closed circles) or chromosomal sequences (green) are used for the introduction of shortened chromosomal arms by homologs recombination (black blocks, telomeric repeats; 1, 2, first/second eukaryotic selectable marker gene). Right: generation of satellite‐based artificial chromosomes (SATACs). Single euchromatin amplification in the centromeric region generates a neocentromere linked to integrated ‘foreign’ DNA (blue) and forms a dicentric chromosome. Breakage between the centromeres generates a chromosome fragment bearing the neocentromere, heterochromatin, and integrated ‘foreign’ DNA. Repeated BrdU treatments and/or drug selection induce further amplification of heterochromatin together with the ‘foreign’ DNA (pink, euchromatin; yellow, heterochromatin).

Figure 3.

Schematic illustration of ‘bottom‐up’ engineered mammalian artificial chromosome (MAC) systems. Left: construction of mini‐EBV‐based MACs. A genomic insert from a bacterial artificial chromosome (BAC) or P1 phage‐derived artificial chromosome (PAC) clone containing the target gene(s) is inserted into the Epstein‐Barr virus (EBV) shuttle vector by Cre–loxP intermolecular recombination (ori, BAC/PAC origin(s) of replication). The EBV shuttle vector contains minimal EBV elements for stable episomal maintenance (rEBNA1, enhanced variant of Epstein–Barr virus nuclear antigen 1 gene, oriP, EBV origin of replication). The BAC/mini‐EBV or PAC/mini‐EBV clone is electroporated in Escherichia coli cells and selected with either chloramphenicol for BAC DNA inserts or kanamycin for PAC DNA inserts. The DNA is purified and transfected into mammalian cells. Transfected cells are selected depending on the introduced eukaryotic selection marker gene (ESM). Right: In order to produce mini‐EBV based MAC virions the EBV packaging signal ‘TR’ is replaced in the wild‐type EBV strain B95.8 with a eukaryotic selectable marker gene (ESM) by homologous recombination. Subsequently, the generated TR‐negative EBV B95.8 plasmid and a mini‐EBV‐based MAC containing ‘TR’ and the target gene(s) are cotransfected into lymphoma helper cells. Following the induction of the lytic cycle, the isolated packaging‐deficient virions are introduced into EBV‐permissive human cells.



Co DO, Borowski AH, Leung JD et al. (2000) Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection. Chromosome Research 8: 183–191.

Ebersole TA, Ross A, Clark E et al. (2000) Mammalian artificial chromosome formation from circular alphoid input DNA does not require telomere repeats. Human Molecular Genetics 9: 1623–1631.

Green LL, Hardy MC, Maynard‐Currie CE et al. (1994) Antigen‐specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nature Genetics 7: 13–21.

Harrington JJ, Van Bokkelen G, Mays RW, Gustashaw K and Willard HF (1997) Formation of de novo centromeres and construction of first‐generation human artificial microchromosomes. Nature Genetics 15: 345–355.

Henning KA, Novotny EA, Compton ST et al. (1999) Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single‐ copy DNA sequences. Proceedings of the National Academy of Sciences of the USA 96: 592–597.

Hodgson JG, Agopyan N, Gutekunst CA et al. (1999) A YAC mouse model for Huntington's disease with full‐length mutant huntington, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23: 181–192.

Ikeno M, Grimes B, Okazaki T et al. (1998) Construction of YAC‐based mammalian artificial chromosomes. Nature Biotechnology 16: 431–439.

Ioannou PA, Amemiya CT, Garnes J et al. (1994) A new bacteriophage P1‐derived vector for the propagation of large human DNA fragments. Nature Genetics 6: 84–89.

Kereso J, Praznovsky T, Cserpan I et al. (1996) De novo chromosome formations by large‐scale amplification of the centromeric region of mouse chromosomes. Chromosome Research 4: 1–14.

Little RD and Schildkraut CL (1995) Initiation of latent DNA replication in the Epstein–Barr virus genome can occur at sites other than the genetically defined origin. Molecular and Cellular Biology 15: 2893–2903.

Mills W, Critcher R, Lee C and Farr CJ (1999) Generation of an approximately 2.4 Mb human X centromere‐based minichromosome by targeted telomere‐associated chromosome fragmentation in DT40. Human Molecular Genetics 8: 751–761.

Mullins LJ, Kotelevtseva N, Boyd AC and Mullins JJ (1997) Efficient Cre‐lox linearisation of BACs: applications to physical mapping and generation of transgenic animals. Nucleic Acids Research 25: 2539–2540.

Murray AW and Szostak JW (1983) Construction of artificial chromosomes in yeast. Nature 305: 189–193.

Nielsen LB, McCormick SP, Pierotti V et al. (1997) Human apolipoprotein B transgenic mice generated with 207‐ and 145‐kilobase pair bacterial artificial chromosomes. Evidence that a distant 5′‐element confers appropriate transgene expression in the intestine. Journal of Biological Chemistry 272: 29752–29758.

O'Connor M, Peifer M and Bender W (1989) Construction of large DNA segments in Escherichia coli. Science 244: 1307–1312.

Probst FJ, Fridell RA, Raphael Y et al. (1998) Correction of deafness in shaker‐2 mice by an unconventional myosin in a BAC transgene. Science 280: 1444–1447.

Saffery R, Irvine DV, Griffiths B et al. (2000) Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore‐associated proteins. Human Molecular Genetics 9: 175–185.

Schlessinger D (1990) Yeast artificial chromosomes: tools for mapping and analysis of complex genomes. Trends in Genetics 6: 248, 255–258.

Sternberg N (1990) Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proceedings of the National Academy of Sciences of the USA 87(1): 103–107.

Velten FW, Renard C, Rogel‐Gaillard C, Vaiman M and Chardon P (1999) Spatial arrangement of pig MHC class I sequences. Immunogenetics 49: 919–930.

Wendelburg BJ and Vos JM (1998) An enhanced EBNA1 variant with reduced IR3 domain for long‐term episomal maintenance and transgene expression of oriP‐based plasmids in human cells. Gene Therapy 5: 1389–1399.

Westphal EM, Sierakowska H, Livanos E, Kole R and Vos JM (1998) A system for shuttling 200‐kb BAC/PAC clones into human cells: stable extrachromosomal persistence and long‐term ectopic gene activation. Human Gene Therapy 9: 1863–1873.

Further Reading

Brown WR, Mee PJ and Hong Shen M (2000) Artificial chromosomes: ideal vectors? Trends in Biotechnology 18: 218–223.

Brune W, Messerle M and Koszinowski UH (2000) Forward with BACs: new tools for herpesvirus genomics. Trends in Genetics 16: 254–259.

Choo KH (2000) Centromerization. Trends in Cell Biology 10: 182–188.

Houdebine LM (2000) Transgenic animal bioreactors. Transgenic Research 9: 305–320.

Komaki S and Vos JM (2000) Epstein‐Barr Virus vectors for gene therapy. Advances in Virus Research 55: 453–462.

Monaco AP and Larin Z (1994) YACs, BACs, PACs and MACs: artificial chromosomes as research tools. Trends in Biotechnology 12: 280–286.

Schindelhauer D (1999) Construction of mammalian artificial chromosomes: prospects for defining an optimal centromere. Bioessays 21: 76–83.

Vos JM (1997) The simplicity of complex MACs. Nature Biotechnology 15: 1257–1259.

Vos JM (1999) Therapeutic mammalian artificial episomal chromosomes. Current Opinion in Molecular Therapy 1: 204–215.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Roosen, Gert, Velten, Florian W, and Vos†, Jean‐Michel H(Mar 2002) Artificial Chromosomes. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0001159]