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Efficient assembly of de novo human artificial chromosomes from large genomic loci

By Joydeep Basu, George Compitello, Gregory Stromberg, Huntington F Willard and Gil Van Bokkelen


BACKGROUND: Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs. RESULTS: We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs) for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100–200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and ß-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection. CONCLUSION: Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors

Topics: Research Article
Publisher: BioMed Central
Year: 2005
DOI identifier: 10.1186/1472-6750-5-21
OAI identifier:
Provided by: PubMed Central

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  1. (1999). Activity of the c-myc replicator at an ectopic chromosomal location. Mol Cell Biol
  2. (1998). Ashlock MA: Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha-satellite DNA and single copy sequences. Proc Natl Acad Sci USA
  3. (2005). Bokkelen G: Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays. Nucl Acids Res
  4. (2003). Cavazzana-Calvo M: LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science
  5. (2001). Cooke HJ: Stable gene expression from a mammalian artificial chromosome. EMBO Rep
  6. (2002). Della Valle G: CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains.
  7. (1978). Eucaryotic DNA: organization of the genome for replication. Cell
  8. (2000). Gene therapy: trials and tribulations. Nat Rev Genet
  9. (1998). GM: Genetic dissection of a mammalian replicator in the human beta-globin locus. Science
  10. (2002). HF: Alpha-satellite DNA and vector composition influence rates of human artificial chromosome formation. Mol Ther
  11. (2004). HF: Analysis of the centromeric regions of the human genome assembly. Trends Genet
  12. (2005). HF: Artificial and engineered chromosomes: Non-integrating vectors for gene therapy. Trends Mol Med
  13. (1997). HF: Formation of de novo centromeres and construction of firstgeneration human artificial microchromosomes. Nat Genet
  14. (2001). HF: Genomic and genetic definition of a functional human centromere. Science
  15. (2003). HF: Human artificial chromosomes with alpha-satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag. Mol Cell Biol
  16. (2000). HJ: Mitotic stability of an episomal vector containing a human scaffold/matrix attached region is provided by association with nuclear matrix. Nat Cell Biol
  17. (2005). Huxley C: Construction of human artificial chromosome vectors by recombineering. Gene
  18. (1997). Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. Hum Mol Genet
  19. (2001). Isolation and subcloning of large fragments from BACs and PACs. Biotechniques
  20. (1987). JS: Chromosome-specific subsets of human alpha-satellite DNA: analysis of sequence divergence within and between chromosomal subsets and evidence for an ancestral pentameric repeat.
  21. (2003). KF: Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell
  22. (2004). KH: Building the centromere: from foundation proteins to 3D organization. Trends Cell Biol
  23. (2003). Larionov V: Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes. Nucleic Acids Res
  24. (2003). Latest development in viral vectors for gene therapy. Trends Biotech
  25. (2000). Mammalian artificial chromosome formation from circular alphoid input DNA does not require telomere repeats. Hum Mol Genet
  26. (2002). Masumoto H: CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA.
  27. (1998). Masumoto H: Construction of YAC-based mammalian artificial chromosomes. Nat Biotechnol
  28. (2002). Mejia JE: Advances in human artificial chromosome technology. Trends Genet
  29. (1968). On the mechanism of DNA replication in mammalian chromosomes.
  30. (1995). PC: The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains. Nat Genet
  31. (1999). Replication origins of mammalian chromosomes: the happy few. Front Biosci
  32. (1998). Reznikoff WS: Tn5 in vitro transposition.
  33. (2001). Silencing of gene expression: implications for design of retrovirus vectors. Rev Med Virol
  34. (2002). Stamatoyannopoulos G: Locus control regions. Blood
  35. (1983). Szostak JW: Construction of artificial chromosomes in yeast. Nature
  36. (2002). T: Generation of human artificial chromosomes expressing naturally controlled guanosine triphosphate cyclohydrolase I gene. Genes Cells
  37. (2000). The assembly of large BACs by in vivo recombination. Genomics
  38. (1993). WR: Telomere directed fragmentation of mammalian chromosomes. Nucl Acids Res
  39. (2001). Z: Functional complementation of a genetic deficiency with human artificial chromosomes.

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