Transgenic zebrafish using transposable elements

Abstract

DNA transposons are effective chromosomal engineering vehicles for making transgenic zebrafish. We describe both autonomous and non-autonomous transposable elements, and we compare and contrast popular transposon systems. The Tol2 system is a robust gene transfer tool and has been selected as the primary transposon platform, facilitating the development of an array of reagents readily shared within the zebrafish community. We present common transposon and transposase vectors within the field based on the Tol2 system. We describe methods with a high success rate of generating transgenic zebrafish using Tol2 vectors, including key quality control steps during the transgenesis process. Together, these data should enable the ready generation of transgenic zebrafish for scientific inquiry.

Figure 1. Transposon structures

A) An autonomous transposon found within the genome in its “wild” configuration contains full inverted terminal repeats (ITR) capable of driving expression of an active transposase located within the ITRs. The transposase is transcribed and processed including polyadenylation from signals located within the ITRs. The transposase mRNA is translated into protein that can recognize sequences at the distal ends of the transposon and “cut” it from the genomic DNA and “paste” it into a new location within the genome. B) Mutations (red areas) in the transposase cause the transposon to become non-autonomous, meaning the transposon has lost its ability to produce functional transposase protein. However, the ITRs of a non-autonomous transposon can be recognized if functional transposase is provided from another source (e.g. an autonomous element elsewhere in the genome). C) An engineered or “domesticated” transposon can be made by replacing the transposase coding region with a different gene or expression cassette. D) As the transposon sequences required for mobilization are understood, a transposon can be made using so-called “minimal” ITR sequences (mITR). Doing so likely removes elements that are required for normal expression of the transposase (promoter, polyadneylation signals, etc.). E) A separate DNA expression cassette can be made by placing the transposase sequence between a promoter (P) and a polyadenylation signal (poly(A)). F) Alternatively, in vitro transcribed mRNA can be produced as a transient source of transposase. In zebrafish work, the most common genetic manipulations include the combination of a minimal transposon (D) and in vitro transcribed mRNA (F).

Figure 2. Overview of Tol2 transgenesis method

The basic approach to producing a transgenic zebrafish using the Tol2 transposon system is diagramed and listed. See text for details.

Figure 3. Available Tol2 transposon vectors

A and B) pDestTol2pA and pDestTol2pA2 are multisite gateway cloning vectors available from the Lawson lab (Villefranc et al., 2007) (http://lawsonlab.umassmed.edu/gateway.html) and the Chien lab (Kwan et al., 2007) (http://chien.neuro.utah.edu/tol2kitwiki/index.php/Main_Page), respectively. They are largely the same although the A2 version removes a large chunk of the O. latipes tyrosinase (tyr) gene that was cloned along with the original Tol2 transposon ITRs. C) pT2AL200R150G is a minimal Tol2 vector available from the Kawakami lab (Urasaki et al., 2006) (http://kawakami.lab.nig.ac.jp/). It includes a simple GFP expression cassette that is removed when cloning a gene between the BglII and XhoI restriction endonuclease sites. D) pminiTol2 is a minimal Tol2 transposon vector, available from the Ekker lab (Balciunas et al., 2006) (http://zfishbook.org), that shares the same multiple cloning sites as many SB vectors derived from pT/HB or pT/BH (Geurts et al., 2003). E) pKTol2-SE is a minimal Tol2 vector with a simplified vector backbone available from the Clark laboratory (http://zfishbook.org). pKTol2-SE shares a multiple cloning site with other vertebrate transposon cloning vectors: pKT2-SE, pPBT-SE, and pPPTn-SE for Sleeping Beauty (Clark et al., 2007; Ivics et al., 1997), piggyBac (Clark et al., 2007; Fraser et al., 1996), and Passport (Clark et al., 2009), respectively. The size of each plasmid (A–E) is noted. In cases where some of the plasmid is removed in the cloning process (A–C), the length of remaining elements is shown in parentheses. Abbreviations: ITR-R (Tol2 inverted terminal repeat right or 3’), ITR-L (Tol2 inverted terminal repeat left or 5’), DEST cassette (required for Gateway cloning, replaced with contents of entry vector), EF1α (elongation factor 1 alpha promoter), RBG intron (rabbit beta-globin intron), EGFP (enhanced green fluorescent protein), SV40(A) (SV40 polyadenylation signal), T7 (T7 polymerase binding site), T3 (T3 polymerase binding site), Lac Promoter (Lac operon promoter), ColE1 ORI (plasmid origin of replication), AmpR (ampicillin resistance gene), KanR (Kanamycin resistance gene), and F1 ORI (single-stranded phagemid origin).

Figure 4. Available Tol2 transposase transcription vectors

A and B) pCS-TP and pCS2-Transposase are very similar transcription vectors available from the Kawakami lab or Chien lab, respectively. Both are based on a pCS vector backbone and use the bacteriophage SP6 polymerase. pCS2-transposase lacks the V1 and V2 regions that correspond to cDNA from the native Tol2 transposase untranslated regions. C) pT3TS-Tol2 is available from the Ekker lab. It uses T3 polymerase to produce mRNA and includes untranslated regions from the Xenopus beta-globin gene. The restriction sites available for linearization of the plasmid are shown in red.