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Development
2024 Aug 15;15120:. doi: 10.1242/dev.202991.
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Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.
Jackson EW
,
Romero E
,
Kling S
,
Lee Y
,
Tjeerdema E
,
Hamdoun A
.
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Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.
Fig. 1. Integrated transgenes are expressed robustly at the late larval and juvenile stages. Transient transgene expression from the reporter construct alone was distinct from genomic transgene expression from bona fide integration at the late larval and juvenile phases. (A) Schematic of the polyubiquitin promoter from L. variegatus used to drive transgene expression. (B) Experimental design for creating F0 sea urchins with somatic integrations that could subsequently be selected for grow out. (C) Transgene expression patterns with and without the Minos transposase over early development. Each data point represents the proportion of offspring with fluorescence from a unique mate pair. In total, 200 larvae were screened for each timepoint from three or four different mate pairs. Data are mean±s.d. Black dashed line represents the transition from larval to juvenile through metamorphosis. *P<0.05 (Welch's two-sample t-test).
Fig. 2. The Minos element resulted in significantly higher somatic integration rates than Tol2, piggyBac or controls.
Tol2 and piggyBac were not significantly different from controls. (A) Each transposon was tested by injecting mRNA of the transposase along with a plasmid containing a polyubiquitin promoter upstream of a human H2B-mCerulean. Control conditions contained only the plasmid. (B) Screening for somatic integration was performed 2-4 days post-metamorphosis. Each data point represents the proportion of offspring with fluorescence from a unique mate pair. Data are mean±s.d. At least five different mate pairs were used for each experimental group (i.e. different transposon tested). ***P<0.0001 (pairwise Wilcoxon rank sum test, Bonferroni-corrected). (C) F0 juveniles injected with the reporter plasmid and with (+) or without (−) the Minos transposase. Juveniles were stained with CellMask plasma membrane orange to create contrast with nuclear CFP signal. Scale bar: 100 μm.
Fig. 3. Mosaicism and variation of transgene expression in F0 animals. Live imaging of nine unique F0 juveniles injected with Minos transposase and our reporter construct. Top and middle rows are aboral views; bottom row is the oral view. Juveniles were stained with CellMask plasma membrane orange to create contrast with nuclear CFP signal. Scale bar: 250 μm.
Fig. 4. Measurement of transgene expression levels by high content screening of F1 embryos. (A) Max fluorescent intensity of transgenic embryos separated by parent with germline integration. Each dot represents one embryo at 24-26 h post fertilization. Box plots show median values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate 1.5× the interquartile ranges; dots indicate data points. (B) Representative image used for machine learning-based automated segmentation to determine integration status and measure CFP fluorescent intensity. The top and bottom images are the same embryos in transmitted light and confocal, respectively. Scale bar: 50 μm.
Fig. 5. Transgenes are robustly and ubiquitously expressed in F1 embryos and larvae through metamorphic competency. Image shows confocal micrographs of F1 embryo and larvae produced from mating wild-type female eggs with sperm of a transgenic male with germline integration. Scale bars: 100 μm.
Fig. 6. Transgene expression in F1 animals is ubiquitous across the body. Live imaging of nine unique F1 juveniles. Top and middle rows are aboral views; bottom row is the oral view. Juveniles were stained with CellMask plasma membrane orange to create contrast with nuclear CFP signal. Scale bar: 250 μm.
