Human Molecular Genetics, Vol 8, 751-761, Copyright © 1999 by Oxford University Press
W Mills, R Critcher, C Lee and CJ Farr
A linear mammalian artificial chromosome (MAC) will require at least three
types of functional element: a centromere, two telomeres and origins of
replication. As yet, our understanding of these elements, as well as many
other aspects of structure and organization which may be critical for a
fully functional mammalian chromosome, remains poor. As a way of defining
these various requirements, minichromosome reagents are being developed and
analysed. Approaches for minichromosome generation fall into two broad
categories: de novo assembly from candidate DNA sequences, or the
fragmentation of an existing chromosome to reduce it to a minimal size.
Here we describe the generation of a human minichromosome using the latter,
top-down, approach. A human X chromosome, present in a DT40-human microcell
hybrid, has been manipulated using homologous recombination and the
targeted seeding of a de novo telomere. This strategy has generated a
linear approximately 2.4 Mb human X centromere-based minichromosome capped
by two artificially seeded telomeres: one immediately flanking the
centromeric alpha-satellite DNA and the other targeted to the zinc finger
gene ZXDA in Xp11.21. The chromosome retains an alpha-satellite domain of
approximately 1. 8 Mb, a small array of gamma-satellite repeat (
approximately 40 kb) and approximately 400 kb of Xp proximal DNA sequence.
The mitotic stability of this minichromosome has been examined, both in
DT40 and following transfer into hamster and human cell lines. In all three
backgrounds, the minichromosome is retained efficiently, but in the human
and hamster microcell hybrids its copy number is poorly regulated. This
approach of engineering well-defined chromosome reagents will allow key
questions in MAC development (such as whether a lower size limit exists) to
be addressed. In addition, the 2.4 Mb minichromosome described here has
potential to be developed as a vector for gene delivery.
ARTICLES
Generation of an approximately 2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40
Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
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