Human mini-chromosomes with minimal centromeres

Abstract

We have introduced a 6.5 Mb human mini-chromosome with a complex centromere structure into DT40 cells and have used sequence targeting and telomere-directed chromosome breakage to dissect the sequence requirements for centromere function. These experiments proved that a vertebrate centromere with two blocks of functional alphoid DNA separated by 2.5 Mb can exist as a stable structure in some but not all vertebrate cells. Further experiments indicated that recovery of chromosomes with less than approximately 100 kb of alphoid DNA is very inefficient, suggesting that a functional centromere requires a minimum of approximately 100 kb of alphoid DNA. Mini-chromosomes with minimal centromeres segregate accurately in some but not all vertebrate cells and should be useful for the detection of sequence-specific factors required for vertebrate centromere maintenance.

Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells

“…The "top-down" approach involves modification of human chromosomes in living cells to produce chromosome derivatives (11)(12)(13)(14)(15)(16)(17)(18). The "bottom-up" approach involves de novo kinetochore assembly from 50-to 100-kb blocks of centromeric alpha-satellite (i.e., alphoid) DNAs.…”

Artificial chromosome formation in maize (Zea mays L.)

“…However, there are several lines of evidence indicating that just a small fraction of the original native centromere can retain centromere function (Maguire 1987;Ananiev et al 1998;Yang et al 2000;Spence et al 2002;Zhong et al 2002;Jin et al 2004;Phelps-Durr and Birchler 2004). Thus, identification of this small fraction of DNA segments that confers centromere function, and kinetochore formation, becomes a critical Fig.…”

“…Fragmentation of mammalian chromosomes by irradiation to produce minichromosomes has been reported (Carine et al 1986), but integration of telomeric DNA and consequent truncation of existing chromosomes has been more widely used, and may result in stable minichromosomes with predictable content and organization. Generation of truncated minichromosomes has been achieved in different human and mammalian cell lines (Heller et al 1996;Kuroiwa et al 1998Kuroiwa et al , 2002aMills et al 1999;Yang et al 2000;Auriche et al 2001;Saffery et al 2001;Katoh et al 2004).…”

Chromosome engineering: prospects for gene therapy

“…More recently, human chromosomes have been transferred into the recombination-proficient chicken cell line DT40, making it practical to target telomere seeding events and generate products of defined structure. [34][35][36] Successive rounds of telomere seeding and chromosome truncation have been used to generate a series of human Y-derived linear minichromosomes ranging in size from approximately 9 Mb down to 1.7 Mb. 35,37,38 While stable in human HT1080 and chicken DT40 cells, when introduced into mouse cells (ES and/or LA-9) various human Y-based minichromosomes were found to be unstable.…”

“…[34][35][36] Successive rounds of telomere seeding and chromosome truncation have been used to generate a series of human Y-derived linear minichromosomes ranging in size from approximately 9 Mb down to 1.7 Mb. 35,37,38 While stable in human HT1080 and chicken DT40 cells, when introduced into mouse cells (ES and/or LA-9) various human Y-based minichromosomes were found to be unstable. 37,39 One of the minichromosomes rearranged and was stabilized through the acquisition of mouse centromeric DNA.…”