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 here ditary 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 th e 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/IPTG(1) agar plat es. Mutations in the repeats characteristic of the RER+-phenotype may result in the loss or gain of CA-repeats leading to blue bacterial col onies. We transiently transfected the colorectal cancer cell lines SW4 80 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 transf ection of the RER+-cell line HCT116, the mutation frequencies increasi ng with incubation time. Sequence analysis of mutated plasmids reveale d mostly 2-bp deletions which occurred especially in one of the repeat tracts. We conclude that pZCA29 appears to be a suitable shuttle vect or for the detection and analysis of a RER+-phenotype in cell lines. ( C) 1998 Elsevier Science B.V.