De Novo Generation of Satellite DNA-Based Artificial Chromosomes by Induced Large-Scale Amplification

Abstract

Mammalian artificial chromosomes (MACs) are engineered chromosomes with defined genetic content that can function as non-integrating vectors with large carrying capacity and stability. The large carrying capacity allows the engineering of MACs with multiple copies of the same transgene, gene complexes, and to include regulatory elements necessary for the regulated expression of transgene(s). In recent years, different approaches have been explored to generate MACs (Vos Curr Opin Genet Dev 8:351–359, 1998; Danielle et al. Trends Biotech 23:573–583, 2005; Duncan and Hadlaczky Curr Opin Biotech 18:420–424, 2007): (1) the de novo formation by centromere seeding, the “bottom-up” approach, (2) the truncation of natural chromosomes or the modification of naturally occurring minichromosomes, the “top-down” approach, and (3) the in vivo “inductive” approach. Satellite DNA-based artificial chromosomes (SATACs) generated by the in vivo “inductive” method have the potential to become an efficient tool in diverse gene technology applications such as cellular protein manufacturing (Kennard et al. BioPharm Int 20:52–59, 2007; Kennard et al. Biotechnol Bioeng 104:526–539, 2009; Kennard et al. Biotechnol Bioeng 104:540–553, 2009), transgenic animal production (Telenius et al. Chromosome Res 7:3–7, 1999; Co et al. Chromosome Res 8:183–191, 2000; Monteith et al. Methods Mol Biol 240:227–242, 2003), and ultimately a safe vector for gene therapy (Vanderbyl et al. Stem Cells 22:324–333, 2004; Vanderbyl et al. Exp Hematol 33:1470–1476, 2005; Katona et al. Cell. Mol. Life Sci 65:3830–3838, 2008). A detailed protocol for the de novo generation of satellite DNA-based artificial chromosomes (SATACs) via induced large-scale amplification is presented.