The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes

Abstract

Tol1 is an active DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes. This element belongs to the hAT transposable element family, of which complete copies have relatively long sequences. In addition, we found that Tol1 elements as long as 18 and 20 kb occur in the medaka fish genome. These facts suggest that Tol1 is suitable for carrying large DNA fragments as a gene transfer vector. Focusing on this, we conducted two kinds of manipulations of the element. The first was to eliminate internal regions dispensable for transposition. It was revealed that a Tol1 element consisting of 157-bp left-and 106-bp right-terminal regions could be transposed without a loss of transposition efficiency. Next, we prepared long Tol1 elements by incorporating unrelated DNA fragments into this short Tol1 clone and examined their transposition efficiencies. The transposition frequency decreased as the element size increased. The longest Tol1 element we examined measured 22.1 kb, and its transposition frequency was approximately one fifth that of a 2.1-kb element. However, this frequency was still significantly higher than that of a random integration of DNA into the chromosomes. The element size of 22.1 kb is the longest ever reported for DNA-based elements currently used for mammals. Thus, Tol1 is a superior genetransfer vector with a large cargo capacity.

Germline transgenesis of zebrafish using the medaka Tol1 transposon system

“…Excisions were imprecise at the nucleotide sequence level, as has previously been observed with Tol1 in medaka (Tsutsumi et al, 2006) and mammals (Koga et al, 2007a).…”

“…A large cargo-capacity and short length of terminal regions necessary for transposition are two key features of Tol1; a Tol1 cassette as long as 22.1 kb and containing a combined Tol1 left and right terminal sequence of only 263 bp, is efficiently transposed from injected plasmids to the chromosome (Koga et al, 2007a). These features are expected to be an advantage for genetic manipulation in zebrafish whose genome size is larger than in medaka and fugu (Lamatsch et al, 2000), and whose gene organization may be larger and structurally more complex or redundant.…”

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

“…These extra nucleotides The deletion of the entire Tol1 region provides clear evidence for the occurrence of excision. The excision was not strictly precise in both samples, as has commonly been observed with Tol1 (Koga et al, 2007b;Hikosaka and Koga, 2007).…”

“…We have previously applied exactly the same procedures to human culture cells (Koga et al, 2007b). On conducting inverse PCR with cells after proliferation through several rounds of cell division, we observed smeared PCR products.…”

“…We have recently found a medaka fish strain that was highly mutable because of high-frequency transposition of the element, and identified an autonomous Tol1 copy by analyzing this strain (Koga et al, 2007a). The autonomous copy carried a transposase gene, and a BLAST search of its amino-acid sequence revealed that Tol1 is an hAT element (Koga et al, 2007b). Tol1 is mobile in many vertebrate species, including the medaka fish (Tsutsumi et al, 2006), zebrafish (manuscript under review), frog (Hikosaka and Koga, 2007), and human and mouse (Koga et al, 2007a).…”