An S/MAR-based L1 retrotransposition cassette mediates sustained levels of insertional mutagenesis without suffering from epigenetic silencing of DNA methylation.
L1 is an insertional mutagen that is capable of mediating permanent gene disruption in mammalian genomes. However, currently available L1 retrotransposition vectors exhibit low or unstable transgene expression when expressed in somatic cells and tissues. This restriction limits their potential utility in long-term screening procedures or somatic mutagenesis applications. In this study, we addressed this problem by developing a minicircle, nonviral L1 retrotransposition vector using a scaffold/matrix attachment region (S/MAR) in the vector backbone and evaluated its utility in human cell lines. The S/MAR-based L1 retrotransposition vector provides stable, elevated levels of L1 expression compared to the currently used EBNA1-based L1 vector. In addition, the S/MAR elements effectively mediate sustained levels of L1 retrotransposition in prolonged cell culturing without suffering from epigenetic silencing by DNA methylation or from vector integration problems even in the absence of selection pressure. These findings indicate that the simple inclusion of S/MAR in the vector backbone increased levels of L1 expression and retrotransposition that can be used as an effective tool to generate insertional mutagenesis in large-scale somatic mutagenesis applications in mammalian cells.
Rangasamy
Danny
D
The John Curtin School of Medical Research, Australian National University, Canberra, Australia. Danny.Rangasamy@anu.edu.au
D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D044127:Epigenesis, Genetic; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D020084:Long Interspersed Nucleotide Elements; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics
D017931:DNA Primers; D019309:Epstein-Barr Virus Nuclear Antigens; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C417029:EBV-encoded nuclear antigen 1