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Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells.
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MedLine Citation:
PMID:  10075999     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.
Authors:
R Wade-Martins; J Frampton; M R James
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Nucleic acids research     Volume:  27     ISSN:  0305-1048     ISO Abbreviation:  Nucleic Acids Res.     Publication Date:  1999 Apr 
Date Detail:
Created Date:  1999-06-09     Completed Date:  1999-06-09     Revised Date:  2010-08-25    
Medline Journal Info:
Nlm Unique ID:  0411011     Medline TA:  Nucleic Acids Res     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1674-82     Citation Subset:  IM    
Affiliation:
The Wellcome Trust Centre for Human Genetics, Nuffield Department of Clinical Medicine, University of Oxford, Windmill Road, Oxford OX3 7BN, UK.
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MeSH Terms
Descriptor/Qualifier:
Cell Line, Transformed
DNA / genetics*
DNA Replication
Flow Cytometry
Humans
Male
Microscopy, Fluorescence
Plasmids*
Transfection*
Grant Support
ID/Acronym/Agency:
//Wellcome Trust
Chemical
Reg. No./Substance:
9007-49-2/DNA

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Nucleic Acids Res
ISSN: 0305-1048
ISSN: 1362-4962
Article Information
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Print publication date: Day: 1 Month: 4 Year: 1999
Volume: 27 Issue: 7
First Page: 1674 Last Page: 1682
ID: 148371
PubMed Id: 10075999
Publisher Item Identifier: gkc292

Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells.
R Wade-Martins
J Frampton
M R James
The Wellcome Trust Centre for Human Genetics, Nuffield Department of Clinical Medicine, University of Oxford, Windmill Road, Oxford OX3 7BN, UK.


Article Categories:
  • Research Article


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