Dataset Information

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

ABSTRACT: 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.

SUBMITTER: Jackson EW

PROVIDER: S-EPMC11361634 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature

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Publications

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Jackson Elliot W EW Romero Emilio E Kling Svenja S Lee Yoon Y Tjeerdema Evan E Hamdoun Amro A

Development (Cambridge, England) 20240819 20

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 ...[more]

PMID: 39023164

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Project description:Using standard morphometric methods and gene expression profiling with a DNA microarray, we explored the impacts of high CO2 conditions on development of the sea urchin, Lytechinus pictus, a pelagic larvae that forms a calcium carbonate endoskeleton. Larvae were raised from fertilization to pluteus stage in seawater with elevated CO2 conditions based upon IPCC emissions scenarios B1 (540ppm CO2) and A1FI (970ppm CO2). Larval L. pictus were cultured in seawater treated with three different concentrations of CO2. We aerated water in culture chambers with air that had been mixed to different CO2 concentrations chosen from IPCC predictions of future atmospheric CO2 levels (28). The B1 scenario, one of the most optimistic, predicts a stabilization of atmospheric CO2 levels at ~540 ppm by 2100, and the A1F1 scenario, which, reflecting a more âbusiness as usualâ scenario, predicts atmospheric CO2 levels of ~970 ppm by 2100. Compressed air (~380 ppm CO2) was used as a control condition. The culture chambers were 20 L buckets medified to allow aeration bubbles of the experimental gas to mix with and gently stir the culture water without contacting the larvae. Replacement seawater (0.35 Î¼m filtered) was added at a constant rate of 0.5 L hr-1. Water pH of each chamber was monitored daily using a Radiometer Analytical PHM240 pH meter. Adult L. pictus were maintained in flowing seawater at ambient temperature (~15-18 ÂºC) and fed kelp until use. Spawning was induced by coelomic injection of 0.5 M KCl, and eggs were collected in 0.35 Î¼m-filtered seawater. Eggs from 5 females were combined in approximately equal concentrations. Sperm from a single male was collected in the same way, diluted with filtered seawater and added to the egg mixture; thus, all the larvae are half-siblings. Immediately following fertilization, embryos were split into 12 culture chambers providing four replicates of the three CO2 treatments. At the 48 hour time point, a sample of 40,000 larvae was removed from each of the 8 cultures in the 380 and 970 ppm treatments and stored in1 mL Trizol at -80 ÂºC for later RNA analysis. The larval cDNA samples from each treatment were competitively hybridized on each microarray following a balanced incomplete block design (BIBD) with respect to the assignment of treatments to arrays. In this way, differential expression between all pairs of treatments is estimated with equal precision. Although dye swaps of technical replicates were not used, treatment and dye effects are orthogonal to one another, i.e. treatment effects can be estimated independently of dye effects.

Dataset Information

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Publications

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

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