Germ-line transgenesis of the Tc1/mariner superfamily transposon Minos in Ciona intestinalis

Abstract

The tadpole larva of the basal chordate Ciona intestinalis has the most simplified, basic body-plan of chordates. Because it has a compact genome with a complete draft sequence, a large quantity of EST/cDNA information, and a short generation time, Ciona is a suitable model for future genetics. We establish here a transgenic technique in Ciona that uses the Tc1/mariner superfamily transposon Minos. Minos was integrated efficiently into the genome of germ cells and transmitted stably to subsequent generations. In addition, an enhancer-trap line was obtained. This is a demonstration of efficient, Minos-mediated transgenesis in marine invertebrates.

Fig. 1.

Inheritance of GFP expression in Ciona F₁ and F₂ progeny. (A) Structure of a Minos construct, pMiLRCiTPO-gfp. The dotted line indicates the probe region used for Southern blot analysis shown in Fig. 2A. Arrows indicate the primer positions used for PCR analysis shown in Fig. 2B. LIR and RIR, left and right inverted repeats (IR) of Minos; pA, polyadenylation signal sequence; EI, EcoRI restriction site. (B–E) F₁ progeny and the expression of GFP from Mi[CiTPOgfp]3 (B and C) and Mi[CiTPOgfp]2 (D and E). In C, GFP was seen at the anterior and posterior ends of the endostyle (red arrows). In E, endostyle (EN), peripharyngeal band (PB), dorsal tubercle (DT), and posterior margin of pharyngeal sac (PM) were marked by GFP. (F and G) GFP expression in F₂ progeny derived from Mi[CiTPOgfp]3 (F) and Mi[CiTPOgfp]2 (G).

Fig. 2.

Genomic integration of Minos revealed by Southern blot and PCR analyses. (A) Genomic DNA isolated from F₁ progeny of Mi[CiTPOgfp]1, 3, and 4 (lanes 1, 3, and 4) and wild type (lane W) were digested with EcoRI and electrophoresed. Right numbers indicate the sizes of markers (in kbp). There are several bands characteristic to each F₁ progeny, in addition to similarly sized bands of ≈1.6 and 2.0 kbp (arrowheads). (B) Several F₁ progeny lacked plasmid sequences of pMiLRCiTPO-gfp flanking Minos IR. Genomic DNA isolated from F₁ progeny from Mi[CiTPOgfp]1–3, -5, -6 (–6), and wild type (W) were subjected to PCR analysis using primers described in Fig. 1 A. pMiLRCiTPO-gfp plasmid (P) was used as a positive control. Lane M indicates the marker. Right numbers show the size of bands (in bp). Note that several F₁ (lanes 1, 3, 4, 10, and 12) lacked left flanking (LF) and right flanking (RF) sequences of IR of pMiLRCiTPO-gfp, although gfp was transmitted. PCR against tyrosinase gene (tyr) was done as an internal control.

Fig. 3.

Examples of genes targeted by Minos and of enhancer-trap events. (A) Genes disrupted by Minos. Exons and introns are shown by boxes and lines, respectively. Exons corresponding to UTRs are labeled in black. Large triangles indicate the insertion sites of Minos. Grail numbers indicate the gene model in the C. intestinalis genome database (http://genome.jgi-psf.org/ciona4). (B–D) Two examples of enhancer-trap events. GFP was expressed in the pharyngeal gill of F₂ progeny derived from Mi[CiTPOgfp]2 (B), but not in Mi[CiTPOgfp]3 (C). (D) A founder animal expressing GFP in the pharyngeal gill. Because the Minos insertion probably occurred in half of the precursor cells of the pharyngeal gill, only half of the gill expressed GFP (left side of the broken line).