The Tol1 transposable element of the medaka fish moves in human and mouse cells

Abstract

DNA-based transposable elements can be used as tools for gene engineering and gene therapy. A great advantage over RNA-mediated elements and retroviruses is the simplicity and safety of usage. The Tol1 element of the medaka fish Oryzias latipes has structural features of DNA-based elements. Although its excision has already been demonstrated, de novo insertion has not been observed, and a transposase has not been hitherto identified. We first cloned, through in silico search alignments and genomic library screenings, a 4.4-kb Tol1 copy carrying open reading frames and then identified, by mRNA analysis, a 2.9-kb transcript coding for 851 amino acids. The protein product of this transcript catalyzed transposition of a nonautonomous Tol1 copy in human and mouse culture cells. This identification of a fully functional Tol1 transposase could lead to the development of new tools for basic and translational molecular biology applications in mammals.

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

“…An important feature of this family is that their complete copies are long in comparison with those of other major families, such as the mariner/Tc1 family (cf. Koga et al 2007). Second, our analysis of the medaka genome revealed the existence of Tol1 copies that are 18 and 20 kb in length, as shown in the initial part of this study.…”

“…We prepared a genomic library of an albino medaka fish exhibiting partial melanin pigmentation in the skin and eyes (Koga et al 2007). This library was used for the selection of Tol1-carrying genomic clones.…”

“…In most cases, the donor plasmids also contained other DNA fragments, such as a marker gene and stuffer fragments. The helper plasmids were the same as those used in our previous study (Koga et al 2007). …”

“…We recently found another element, Tol1 of the medaka fish, which has the potential for development as a genetic tool. The Tol1 element with an embedded neomycin-resistance gene was efficiently transposed into the chromosomes of human and mouse cells, conferring neomycin resistance on the cells (Koga et al 2007).…”

“…We then constructed Tol1 elements of various lengths by inserting extra DNA fragments into the basic vector and measured their transposition frequencies. As previously described (Koga et al 2007), the Tol1 DNAs were introduced into cells by the lipofection method, and the transposition frequency was determined by counting surviving colonies after selective culturing with G418. Because the efficiency of DNA intake by lipofection is affected by DNA size, we took a measurement to compensate for the size effect: comparisons were made among plasmids of a defined total size that carried Tol1 elements of different sizes.…”