Predictable mosaic transgene expression in ascidian embryos produced with a simple electroporation device

Abstract

Two customized electroporators were specifically designed for creating transgenic ascidian embryos. These electroporators were simple to build, inexpensive, and produced transgenic embryos with efficiencies that equaled or rivaled commercially available machines. A key design feature of these machines resulted in the generation of consistent electroporation pulses providing repeatability between experiments. These devices were used to optimize experimental parameters allowing for the creation of transient transgenic embryos with predictable patterns of mosaic transgene expression. We used these new electroporators to examine the expression of two different fluorescent protein reporter genes with regard to embryonic cell lineage. In general, transgene expression followed the embryonic cell lineage and coelectroporated transgenes were always expressed in the same embryonic cells. Our analysis also indicated that, during development, transgenes could be lost from embryonic cells, suggesting that transgenes may be present in extrachromosomal arrays, as has been observed in other organisms. Our new electroporator designs will allow ascidian researchers to inexpensively produce transgenic ascidians and should prove useful for adapting the electroporation technique to other marine embryo systems. Developmental Dynamics 235: 1921-1932, 2006.

Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9

“…A cleavage assay is likely to underestimate actual mutagenesis efficiency due to mosaicism of electroporated plasmids in Ciona embryos (Zeller et al, 2006). Electroporated embryos contain both transfected and non-transfected cells.…”

miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway

“…Each of these transgenes was driven in the larval epidermis using an epidermis-specific enhancer of the Ciona EpiB gene. Exactly 8 g of each of the three transgenes was electroporated into experimental embryos (Zeller et al, 2006a). For negative control embryos, a neutral YFP transgene was electroporated in place of the miR-124-expressing transgene as a mass control.…”

Ascidians: an invertebrate chordate model to study Alzheimer’s disease pathogenesis

“…Culturing and electroporation of Ciona intestinalis embryos has been described elsewhere (Zeller, 2004;Zeller et al, 2006a). Adult ascidians were collected under permit from several marinas in San Diego, California and maintained in a closed, recirculating seawater system under constant light to induce gamete production.…”

“…Our lab has developed an efficient electroporation-based method for generating hundreds of transgenic ascidian embryos (Zeller, 2004;Zeller et al, 2006a). Fertilized ascidian eggs are mixed with supercoiled plasmid DNA, subjected to an electrical pulse and allowed to develop.…”

“…Tissue-specific misexpression of wild-type or mutant proteins can be achieved by using promoter elements to drive the expression of any protein of interest. We have carefully analyzed and optimized the experimental conditions, and have shown that co-electroporated transgenes are expressed in the same cells (in >95% of embryos examined) and that the electroporation conditions can be varied to adjust the level of transgene expression in a predictable manner (Zeller et al, 2006a). However, to date, there has been no concerted effort to exploit ascidian embryos as a model for a specific human disease.…”