PMID: 9637240Jun 24, 1998Paper

A novel plasmid shuttle vector for the detection and analysis of microsatellite instability in cell lines

Mutation Research
C Diem, T M Rünger

Abstract

Microsatellite instability is an important feature of tumors from hereditary nonpolyposis colorectal carcinoma (HNPCC) patients as well as a variety of sporadic tumors. Here, we present a novel plasmid shuttle vector for the detection of this replication error (RER+) phenotype in human cell lines. The episomely replicated plasmid pZCA29 harbours the bacterial beta-galactosidase gene interrupted by two palindromically arranged poly-(CA)-repeat tracts. The resulting + 1-frameshift leads to white colonies of Escherichia coli DH10B on X-Gal/IPTG1 agar plates. Mutations in the repeats characteristic of the RER+-phenotype may result in the loss or gain of CA-repeats leading to blue bacterial colonies. We transiently transfected the colorectal cancer cell lines SW480 and HCT116 with the plasmid pZCA29, isolated replicated plasmid DNA after several days and used it to transform E. coli DH10B. We found 1.0 to 1.7% blue colonies after passage of the plasmid through the RER+-cell line SW480 in contrast to 3.5 to 8.1% blue colonies after transfection of the RER+-cell line HCT116, the mutation frequencies increasing with incubation time. Sequence analysis of mutated plasmids revealed mostly 2-bp deletions which occurred especially in one of...Continue Reading

References

Sep 1, 1992·Mutation Research·H Sies, C F Menck
Jun 1, 1992·Molecular and Cellular Biology·S T Henderson, T D Petes
May 1, 1992·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·T A Kunkel
Oct 1, 1986·Molecular and Cellular Biology·M Protić-SabljićK Dixon
Oct 24, 1995·Proceedings of the National Academy of Sciences of the United States of America·M StrandT D Petes
Jun 30, 1995·Science·N PapadopoulosK W Kinzler
Nov 4, 1994·Science·A UmarT A Kunkel
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May 7, 1993·Science·L A AaltonenS R Hamilton
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Oct 1, 1995·Trends in Biochemical Sciences·R D Kolodner

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