Engineering mammalian chromosomes

Brenda Grimes, Howard Cooke

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic α-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homogeneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that α-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.

Original languageEnglish (US)
Pages (from-to)1635-1640
Number of pages6
JournalHuman Molecular Genetics
Volume7
Issue number10
DOIs
StatePublished - 1998
Externally publishedYes

Fingerprint

Mammalian Chromosomes
Centromere
Mammalian Artificial Chromosomes
Telomere
Chromosomes
DNA
Transfection
Yeast Artificial Chromosomes
Satellite DNA
Replication Origin
Nucleic Acid Repetitive Sequences
Genes
Seeds
Maintenance
Cell Line

ASJC Scopus subject areas

  • Genetics

Cite this

Engineering mammalian chromosomes. / Grimes, Brenda; Cooke, Howard.

In: Human Molecular Genetics, Vol. 7, No. 10, 1998, p. 1635-1640.

Research output: Contribution to journalArticle

Grimes, Brenda ; Cooke, Howard. / Engineering mammalian chromosomes. In: Human Molecular Genetics. 1998 ; Vol. 7, No. 10. pp. 1635-1640.
@article{6eba6b69aa604501a4dba171335f2807,
title = "Engineering mammalian chromosomes",
abstract = "Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic α-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homogeneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that α-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.",
author = "Brenda Grimes and Howard Cooke",
year = "1998",
doi = "10.1093/hmg/7.10.1635",
language = "English (US)",
volume = "7",
pages = "1635--1640",
journal = "Human Molecular Genetics",
issn = "0964-6906",
publisher = "Oxford University Press",
number = "10",

}

TY - JOUR

T1 - Engineering mammalian chromosomes

AU - Grimes, Brenda

AU - Cooke, Howard

PY - 1998

Y1 - 1998

N2 - Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic α-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homogeneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that α-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.

AB - Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic α-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homogeneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that α-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.

UR - http://www.scopus.com/inward/record.url?scp=0031665677&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031665677&partnerID=8YFLogxK

U2 - 10.1093/hmg/7.10.1635

DO - 10.1093/hmg/7.10.1635

M3 - Article

VL - 7

SP - 1635

EP - 1640

JO - Human Molecular Genetics

JF - Human Molecular Genetics

SN - 0964-6906

IS - 10

ER -