Zeller Robert W, Virata Michael J, Cone Angela C
Molecular Biology Institute and Coastal and Marine Institute, San Diego State University, San Diego, California 92182-4614, USA.
Dev Dyn. 2006 Jul;235(7):1921-32. doi: 10.1002/dvdy.20815.
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.
专门设计了两台定制的电穿孔仪用于创建转基因海鞘胚胎。这些电穿孔仪制作简单、成本低廉，所产生的转基因胚胎效率与市售仪器相当或可与之媲美。这些仪器的一个关键设计特点是能产生一致的电穿孔脉冲，从而在实验之间提供可重复性。这些装置用于优化实验参数，以便创建具有可预测的镶嵌转基因表达模式的瞬时转基因胚胎。我们使用这些新的电穿孔仪来研究两种不同荧光蛋白报告基因在胚胎细胞谱系方面的表达。一般来说，转基因表达遵循胚胎细胞谱系，共电穿孔的转基因总是在相同的胚胎细胞中表达。我们的分析还表明，在发育过程中，转基因可能会从胚胎细胞中丢失，这表明转基因可能存在于染色体外阵列中，正如在其他生物体中所观察到的那样。我们新的电穿孔仪设计将使海鞘研究人员能够低成本地生产转基因海鞘，并且应该证明对将电穿孔技术应用于其他海洋胚胎系统是有用的。
