Transgenic cell lines are models for study of the repeat instability

Abstract

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The expansion of repeats is a type of mutation, characterized by an increase in the number of repeats copies in genomic DNA. This phenomenon leads to a certain human genetic diseases, such as Huntington's disease, Fragile X syndrome, ALS, myotonic dystrophy, several types of spinocerebellar ataxia. More than 30 diseases are caused by expansions of repeats dispersed throughout the human genome. The pathogenesis of most of diseases has been studied in detail, however today the cause of expansion is unknown. There are a number of hypotheses describing possible models for the development of mutation. However these assumption are not supported by experimental data. The aim of our study was to create a cellular model that would allow to evaluate the effect of DNA metabolism in the cell, in particular, transcription and transcription coupled repair, on the (CGG)n instability. CGG-repeat expansion in 5'UTR of FMR1 gene associated with Fragile X syndrome - the main cause of hereditary mental retardation. As a result, we have produced a transgenic cell line based on HEK293A cells as such a model. First of all, we synthesized DNA fragments containing extended CGG-repeat, amplified by GC-rich PCR. Two types of plasmids were used for integration into the genome. In vectors under the inducible promoter TRE. DNA fragments carrying the CGG-repeat and the open reading frame of gFp were inserted. Also this vector encoded the dsRe-dExpress protein with the constitutive promoter regulation which is necessary for determining the efficiency of transfection and selection of transformed cells. Thus, we obtained several types of plasmids carrying a CGG-repeat of various lengths. After transfection, cells were selected using flow cytofluorimetry. The obtained cell lines were thoroughly characterized: the sites of plasmid integration into the genome, the expression levels of fluorescent proteins, and the changes in (CGG)n repeat length within several months of cultivation upon induction of the TRE promoter were determined. Thus, cell lines with controlled transcription across repeat have been produced. Its makes possible to assess the effect of transcription on the repeat expansion. Detailed characterization allows tracking all changes in the repeat region. The obtained cell lines can be used as a model to study the effect of transcription on the repeat instability that is prone to expansion.