De novo retrotransposition of unbiased sequences in a human breast cancer cell clone

It has been demonstrated recently that certain repetitive sequences and eve n expressed single-copy genes are capable of retrotransposition, but little is known about the endogenous or exogenous modifiers of this process in hu man cells. Retrotransposition may contribute to gene inactivation and genet ic instability in cancer development. We have used the human cell line MCF- 7 to generate a method for investigating de novo retrotransposition in brea st cancer cells. The strategy employs a reporter construct transfected into MCF-7 cells that encodes neomycin phosphotransferase gene (neoR) sequences interrupted by an intron derived from the gamma-globin gene and sandwiched between Mo promoters in opposite orientation; the phosphotransferase is no t produced in transfected cells expressing the plasmid until transposition via a spliced antisense neoR RNA intermediate has occurred, conferring a fu nctional gene product and thereby resistance to G418, A stable transfectant line that showed presence of reporter plasmid DNA and expression of report er antisense neoR was obtained and used to demonstrate spontaneous retrotra nsposition of neoR sequences: tester cells were subjected to selection in G 418 medium, and neomycin-resistant clones were isolated at a frequency of 1 0(-7). A Simple PCR-based prescreening of colonies fixed and stained in Pet ri dishes can be used to verify intronless neoR DNA. Expanded populations o f G418-resistant colonies were determined to be derived from reporter seque nces that had transposed via an RNA intermediate by Southern blot genotypin g. This experimental assay may be used for exploring endogenous and environ mental factors that influence host cell-mediated retrotransposition of unbi ased cellular sequences in breast tumor cells. (C) 1999 Wiley-Liss, Inc.