Figure 1.
Excision of a microinjected Tol2-based construct.
(A) Scheme of donor vector pTol2{rps9::egfp}frkt868; the rps::egfp cassette is flanked by inverted Tol2 repeats (Tol2IR) that serve as recognition site for Tol2 transposase co-injected as mRNA along with the vector DNA. (B, C) PCR using vector-specific primers (black arrows) yields a 200 bp PCR fragment specifically from embryos co-injected with tol2 transposase mRNA (left lane), but not from controls (right lane). (D) Precise cleavage of the reporter construct at the end of the Tol2 IRs is evidenced by sequencing of the 200 bp fragment.
Figure 2.
Excision of a microinjected Mos-based construct.
(A) Scheme of donor vector pMos{rps9::egfp}frkt1074; in analogy to pTol2{rps9::egfp}frkt868 (Figure 1A), the rps::egfp cassette is flanked by inverted Mos repeats (MosIR) that serve as recognition site for Mos1 transposase. (B, C) PCR using vector-specific primers (black arrows) yields a variety PCR fragment from embryos co-injected with mos1 transposase mRNA (left lanes), but not controls (right lanes). (D) Sequencing of 21 individual PCR product reveals imprecise breakpoints caused by impaired excision of reporter constructs and/or subsequent modification of DNA ends in the context of non-homologous end repair. See Alignments S1 and S2 for details.
Figure 3.
Tol2-mediated transgenesis with pTol2{rps9::egfp} yields robust, ubiquitous expression of EGFP in G0 animals.
(A, B) pTol2{rps9::egfp}frkt868-injected animals co-injected with tol2 mRNA (A) show significantly stronger EGFP fluorescence (green arrows) than those without transposase (B) (ae: autofluorescence around the adult eyes). (C, D) Stable ubiquitous EGFP expression (green arrows) two months after co-injection of donor DNA and tol2 mRNA (C) compared to a non-injected animal (D). Labels indicate autofluorescence of the jaws (j) as well as the iridophore pigments (p) in both the head and trunk.
Figure 4.
Genomic integration and inheritance of Tol2-mediated constructs.
(A) Genomic Southern blot with an egfp probe reveals genomic integrations of pTol2{rps9::egfp}frkt868 in tested G0 individuals (arrows). (B) Precise genomic integration of a the reporter construct at the end of the IR as revealed by sequencing of an amplicon including neighboring genomic DNA, generated using Tol2IR-specific TAIL-PCR primers. (C) Integration and inheritance of egfp in individual G0 and G1 animals: PCR amplicons specific for egfp (arrowheads) can be detected in genomic DNA of both G0 (lanes 9–18) and G1 (lanes 1–8) individuals. Controls: no DNA (lane 19), wild-type gDNA (lane 20), gDNA extracted from a tuba::egfpvbci1 transgenic individual (lane 21), reporter construct plasmid DNA (lane 22).
Table 1.
Efficiency of Tol2 and Mos1 transposon-mediated transient transgenesis.
Figure 5.
The maf locus drives EGFP in putative neurosecretory brain cells
. (A) Scheme of donor vector pTol2{maf::egfp}frkt1208 containing a 3.6 kB upstream of the maf start codon (compare to Figure 1A). (B) Expression of endogenous maf RNA (arrowhead) in the medial central brain of early metatrochophore larvae. (C) Synexpression of the insulin-like peptide 2/ilp2 mRNA in the same region (arrowhead), suggesting that maf demarcates neurosecretory cells. (D–F) EGFP expression driven by pTol2{maf::egfp}frkt1208 demarcates cells in the same region as endogenous maf.
Figure 6.
EGFP expression at the trochophore stage reflects endogenous tuba expression in tuba::egfpvbci1 animals.
(A) Scheme of donor vector pMos{tuba::egfp}frkt707 containing 4.4 kB of DNA upstream of the alpha-tubulin (tuba) start codon (compare to Figure 2A). (B, C) Expression of endogenous tuba in the prototroch (pt; arrows), apical ciliary tuft (at) and paratrochs (pat; arrowheads) of Platynereis larvae at the trochophore (B) and metatrochophore (C) stages. (D, E) Expression of egfp RNA (D) and EGFP protein in tuba::egfpvbci1 trochophore larvae perfectly reflects endogenous tuba expression. (F) egfp expression in tuba::egfpvbci1 metatrochophore larvae remains restricted to the prototroch (arrow); (G) Stable inheritance of the construct, as evidenced by Southern blots of carriers from three different generations, probed with a labeled egfp fragment.
Table 2.
Survival and transmission rates for the injection of Mos1-based fluorescent reporter constructs.