Abstract
BackgroundAlthough most of long interspersed elements (LINEs), one class of non-LTR-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene (rDNA) clusters, telomeric repeats and other repetitive sequenes. Most of the sequence-specific LINEs have been reported mainly among invertebrate species and shown to retrotranspose into the specific sequences in vivo and in vitro systems. Recenlty, 28S rDNA-specific LINE R2 elements are shown to be distributed among widespread vertebrate species, but the sequence-specific retrotransposition of R2 has never been demonstrated in vertebrates.ResultsHere we cloned a full length unit of R2 from medaka fish Oryzias latipes, named R2Ol, and engineered it to a targeted gene integration tool in zebrafish. By injecting R2Ol-encoding mRNA into zebrafish embryos, R2Ol retrotransposed precisely into the target site at high efficiency (98%) and was transmitted to the next generation at high frequency (50%). We also generated transgenic zebrafish carrying the enhanced green fluorescent protein (EGFP) reporter gene in 28S rDNA target by the R2Ol retrotransposition system.ConclusionsSequence-specific LINE retrotransposes into the precise sequence using target primed reverse transcription (TPRT), possibly providing an alternative and effective targeted gene knockin method in vertebrates.
Highlights
Most of long interspersed elements (LINEs), one class of non-Long terminal repeat (LTR)-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene clusters, telomeric repeats and other repetitive sequenes
Two constructs mutated at the essential amino acid sites for EN and reverse transcriptase (RT) domains, completely abolishing the retrotransposition activities (Fig. 1b, (2) R2 from Oryzias latipes (R2Ol) Reverse transcriptase mutant (RTmut) and (3) R2Ol Endonuclease domain mutant (ENmut)), suggesting that R2Ol moves by target primed reverse transcription (TPRT) and not by recombination events
We further analyzed the 5′-junctions between the retrotransposed R2Ol and the 28S ribosomal DNA (rDNA) target using four Open reading frame (ORF) primers sets (Fig. 1a, blue primers)
Summary
Most of long interspersed elements (LINEs), one class of non-LTR-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene (rDNA) clusters, telomeric repeats and other repetitive sequenes. The molecular mechanism of donor DNA template-dependent HDR requires further elucidation [3], and off-target concerns [10] as well as cancer risk [11, 12] should be monitored. Another system independent of HDR might provide an alternative solution for efficiently targeted gene knockin
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