Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon

Abstract

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.

Pig transgenesis by Sleeping Beauty DNA transposition

“…Although this aspect is rarely experimentally addressed in reports describing transposon-directed gene delivery, methods for co-delivery of transposon donor plasmid with in vitro-synthesized mRNA encoding the transposase to somatic cells have been described (Wilber et al 2006) and may be applicable in protocols of SB-directed pig transgenesis. Also, the use of in vitro-transcribed mRNA as a source of transposase has been successful in early embryos of frog (Sinzelle et al 2006), zebrafish (Davidson et al 2003), and mouse (Dupuy et al 2002).…”

“…The element has been explored for the use in therapeutic gene transfer (Izsvak and Ivics 2004;Liu et al 2006;Mates et al 2009;Mikkelsen et al 2003;Ohlfest et al 2004;Singh et al 2008;Yant et al 2000) and is widely used as a tool in animal transgenesis. SB-directed transgenesis is well-established in zebrafish (Davidson et al 2003), and germline mutagenesis by microinjection of transposon DNA and transposaseencoding nucleic acids into fertilized oocytes has been established in the mouse (Dupuy et al 2002;Mates et al 2009) and the frog (Sinzelle et al 2006;Yergeau et al 2009). …”

A flk‐1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: An in vivo model for vascular studies

“…SB has been used to deliver genes into the germline of mice, fish, and Xenopus (Dupuy et al, 2002;Davidson et al, 2003;Grabher et al, 2003;Sinzelle et al, 2006). It has also been used as a gene therapy vector in the treatment of mouse models of human genetic diseases (Hackett et al, 2005).…”

Trans-Kingdom Transposition of the Maize Dissociation Element

“…It has been generally assumed that a transposable element should have a higher chance for successful transfer in taxonomically close species. Partly driven by this assumption, traditionally only transposons of animal origin were utilized in heterologous animals (Ivics et al 1997;Fadool et al 1998;Raz et al 1998;Kawakami et al 2000;Fischer et al 2001;Horie et al 2001;Davidson et al 2003;Ding et al 2005) and plant transposons were used exclusively in plants (Haring et al 1991). To date, none of the known elements was successfully used in both plant and animal hosts.…”