Trans-Kingdom Transposition of the Maize Dissociation Element

Abstract

Transposons are very valuable tools for genetic manipulation. However, the number of transposable elements that have been suitably adapted for experimental use is insufficient and the spectrum of heterologous hosts in which they have been deployed is restricted. To date, only transposons from animal hosts have been utilized in heterologous animal species and transposons of plant origin have been used in plant genetics. There has been no experimental evidence that any of the known elements could transpose in hosts belonging to both kingdoms. Here we demonstrate that the maize Dissociation (Ds) element is capable of effective Activator (Ac) transposase-mediated transposition in the zebrafish Danio rerio, yielding remarkable germline transmission rates. In addition, mammalian cells were also found to be conducive to Ds transposition. Furthermore, we demonstrate that nuclear localization of Ac transposase is essential for genomic Ds transposition. Our results support the hypothesis that Ac/Ds elements do not rely on hostspecific factors for transposition and that host factors involved in their mobility mechanism are widely conserved. Finally, even in vertebrate cells, the Ac/Ds system displays accurate transposition, largefragment carrying capacity, high transposition frequencies, efficient germline transmission, and reporter gene expression, all of which are advantageous for various genetic applications and animal biotechnology.

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome

“…FlipTraps thus open up the potential for Cre conditional alleles in any species with exons (which includes most eukaryotes) and in which a transposon will work. The Tol2 and Ac transposons have already been shown to mobilize in species ranging from Drosophila (Urasaki et al 2008), zebrafish, and Xenopus to chickens, mice, and humans (Emelyanov et al 2006;Kawakami 2007;Sato et al 2007).…”

“…The Tol2 and maize Ac/Ds transposable systems were chosen to integrate the vector into the genome because of their reported high insertional rate, large cargo capacity, ease of use, and functionality in a wide spectrum of heterologous hosts (Weil and Kunze 2000;Kawakami et al 2004;Balciunas et al 2006;Emelyanov et al 2006). The internal exon is devoid of initiation and stop codons and is flanked by splice acceptor and donor sequences from the zebrafish ras-associated domain family 8 (rassf8) gene, which contains a naturally occurring large internal exon and is constitutively spliced (Fig.…”

“…The fused FlipTrap fragment was subsequently cloned into either the T2KXIGd-in (Kawakami et al 2004) or pMDS-eGFP (Emelyanov et al 2006) at XhoI/BlgII and XhoI/SnaBI, respectively. The full sequence of the FlipTrap vector is available through NCBI (accession no.…”

Dynamic microtubules at the vegetal cortex predict the embryonic axis in zebrafish

“…Transgenic zebrafish lines expressing the following microtubule markers were used for live imaging: (1) Tg(ef1:dclk-GFP) transgene contains two doublecortin domains (microtubule-binding domain) of zebrafish dclk1 fused to gfp, and under the control of the Xenopus ef1 promoter; (2) Tg(zpc:Mmu.Eb1-GFP) fish was generated by injecting a Tol2 plasmid containing a part of zona pellucida protein C promoter (Onichtchouk et al, 2003), and murine EB1-GFP coding sequences (Mimori-Kiyosue et al, 2000), together with Tol2 transposase mRNA into 1-cell stage embryos (Kawakami et al, 2004); (3) Tg(Xen.ef1:Hsa.EB3-GFP) fish were generated by injecting a Ds plasmid containing the Xenopus ef1 promoter (Johnson and Krieg, 1994) and human EB3-GFP sequences (Stepanova et al, 2003), together with Ac transposase mRNA into 1-cell stage zebrafish embryos (Emelyanov et al, 2006).…”

Transposon tools hopping in vertebrates

“…Most recently, the possibility of employing a plant transposon in a vertebrate was exploited. A chimeric Ac transposase (NLS-Tpase), composed of an animalorigin nuclear localization signal (NLS) fused to the N-terminal of the truncated maize Ac lacking the fist 102 amino acids (Tpase 103-807), demonstrated Ds element transposition in zebrafish in both somatic and germline cells [78]. This modified Ac (NLS K5E-Tpase) effectively inserts Ds element DNA in a human embryonic kidney cell line as well [78].…”