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Fig. 1 | Mobile DNA

Fig. 1

From: Targeted gene knockin in zebrafish using the 28S rDNA-specific non-LTR-retrotransposon R2Ol

Fig. 1

R2Ol retrotransposition assay in zebrafish. a Left panel: R2Ol ORF is indicated by a gray closed rectangle and the 5′ and 3′ untranslated region (UTR) by horizontal lines. The approximate positions of the reverse transcriptase (RT) and endonuclease (EN) are indicated. Vertical lines represent the cysteine-histidine motifs. Horizontal arrows indicate the primers used to detect retrotransposition events. The rDNA unit is indicated by a black closed rectangle. The double-strand sequences of the precise insertion sites are indicated. The bottom and putative top strand cleavage sites are indicated by solid and dashed line, respectively. In the downstream 28S sequence of R2Ol, 1 to 4 (TAGC) and 1 to 10 (TAGCCAAATG) nucleotides are denoted as r4 and r10, respectively, and shown by red letters and horizontal arrows. Right panel: A schematic diagram of the R2Ol ribonucleoprotein (RNP) complex and target primed reverse transcription (TPRT) mechanism. R2Ol mRNA (red) anneals with the target DNA (28S rDNA) at the 3′ junction to initiate cDNA (blue) synthesis. b Schematic diagram of the R2Ol mRNA constructs used in the retrotransposition assay. T7, T7 promoter; red box, 28S rDNA; green line, vector (v) sequence; (2) R2Ol RTmut, RT mutant of FADD- > FAYD and (3) R2Ol ENmut, EN mutant of IPD- > IPA (mutation indicated by the dashed line). F0 retrotransposition efficiency for each construct are indicated on the right (see also Table 1). c 3′ and 5′ junction PCR assay of inserted R2Ol for retrotransposition detection. Right panel: 3′ junction PCR results of R2Ol-r4-v. P, positive-control PCR with medaka fish genomic DNA; N, negative-control PCR with uninjected zebrafish genomic DNA; 1–12, injected samples. 3′ junction sequence of the R2Ol retrotransposition events showed in the bottom. Green indicates R2Ol and black indicates 28S rDNA sequences. Left Panel: 5′ junction analysis of inserted R2Ol-r4-v. Green boxes indicate vector sequences, respectively. Non-templated indicates extra sequences at the junction derived from neither 28S rDNA nor R2Ol. Truncated or deleted sites are indicated by nucleotide numbers. The clone number of each insertion type is indicated in the far-right column

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