RRC ID 58022
Author Su Y, Nichuguti N, Kuroki-Kami A, Fujiwara H.
Title Sequence-specific retrotransposition of 28S rDNA-specific LINE R2Ol in human cells.
Journal RNA
Abstract R2 is a long interspersed element (LINE) found in a specific sequence of the 28S rDNA among a wide variety of animals. Recently, we observed that R2Ol isolated from medaka fish, Oryzias latipes, retrotransposes sequence specifically into the target sequence of zebrafish. Because the 28S target and flanking regions are widely conserved among vertebrates, we examined whether R2Ol can also integrate in a sequence-specific manner in human cells. Using adenovirus-mediated expression of R2Ol constructs, we confirmed an accurate insertion of R2Ol into the 28S target of human 293T cells. However, the R2Ol mutant devoid of endonuclease (EN) activity showed no retrotransposition ability, suggesting that the sequence-specific integration of R2Ol into 28S rDNA occurs via the cleavage activity of EN. By introducing both R2Ol helper virus and donor plasmid in human cells, we succeeded in retrotransposing an exogenous EGFP gene into the 28S target site by the trans-complementation system, which enabled simplification of specific gene knock-in in a time-efficient manner. We believe that R2Ol may provide an alternative targeted gene knock-in method for practical applications such as gene therapy in future.
Volume 25(11)
Pages 1432-1438
Published 2019-11-1
DOI 10.1261/rna.072512.119
PII rna.072512.119
PMID 31434792
PMC PMC6795142
MeSH Adenoviridae / genetics DNA, Ribosomal / genetics* Gene Knock-In Techniques Genetic Complementation Test HEK293 Cells Helper Viruses / genetics Humans Long Interspersed Nucleotide Elements* Plasmids RNA, Ribosomal, 28S / genetics* Retroelements*
IF 4.32
Times Cited 0
Human and Animal Cells 293T(RCB2202)