A novel plasmid shuttle vector for the detection and analysis of microsatellite instability in cell lines.
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 the repeat tracts. We conclude that pZCA29 appears to be a suitable shuttle vector for the detection and analysis of a RER+-phenotype in cell lines.
Diem
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Department of Dermatology, University of Würzburg, Germany.
Rünger
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TM
D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D016368:Frameshift Mutation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012107:Research Design; D013997:Time Factors; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured