Gyula Hadlaczky (ed.),
Mammalian Chromosome Engineering: Methods and Protocols
, Methods in Molecular Biology, vol. 738,DOI 10.1007/978-1-61779-099-7_7, © Springer Science+Business Media, LLC 2011
Construction and Use of a Bottom-Up HAC Vector for Transgene Expression
Masashi Ikeno and Nobutaka Suzuki
Recent technological advances have enabled visualization o the organization and dynamics o localchromatin structures; however, the global mechanisms by which chromatin organization modulates generegulation are poorly understood. We designed and constructed a human articial chromosome (HAC) vector that allows regulation o transgene expression and delivery o a gene expression platorm intomany vertebrate cell lines. This technology or manipulating a transgene using a HAC vector could beused in applied biology.
Alphoid DNA, BAC, HAC, MMCT, Cre/lox recombination, Transgene
A human articial chromosome (HAC) is a mini-chromosomethat is constructed articially in human cells (1, 2). Using its ownsel-replicating and segregating systems, a HAC can behave as astable chromosome that is independent rom the chromosomeso host cells. HACs were constructed using a bottom-up strategy based on the transection o cloned or synthetic centromericalphoid DNA precursors with CENP-B boxes into a culturedhuman cell line, HT1080 (3–5). The HACs were built up tomegabase size (1–10 Mb) by multimerization o alphoid precur-sors. This “bottom-up construction” strategy involves the de novoconstruction o HACs by introducing DNA elements necessary or the maintenance o chromosome unction into cells. By con-trast, “top-down construction” reers to the truncation o naturalchromosomes into smaller sizes by using targeting vectors con-taining telomeric sequences (6, 7).