Artificial Chromosomes

Abstract

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.

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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.

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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.

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