Permeabilization of Drosophila embryos for introduction of small molecules.
Permeabilization of Drosophila embryos for introduction of small molecules.
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DOI：
10.1016/j.ibmb.2010.07.007
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发表时间：
2010-11
影响因子：
3.8
通讯作者：
Clason, Todd
中科院分区：
文献类型：
--
作者：
Rand, Matthew D.;Kearney, Alison L.;Dao, Julie;Clason, Todd
关键词：
果蝇胚胎的药理操作因蛋壳的不透过性而受阻。小分子溶质输送到胚胎的最终障碍是位于外部绒毛膜层下面的蜡状层，它包裹着蛋壳下面的卵黄膜。传统的方案需要庚烷或辛烷来渗透脱氯蛋壳，然而，这些溶剂是有毒的，可能导致低生存能力。此外，正庚烷和辛烷要求胚胎在水相和有机相溶剂之间过渡，这使得避免干燥具有挑战性。在这里，我们描述了一种胚胎渗透溶剂(EPS)，它由D-柠檬烯和植物来源的表面活性剂组成，是水可溶的，能高效地使脱氯蛋壳变得可渗透。EPS透过性使胚胎能够吸收几种不同分子质量的不同染料，最高可达995道尔顿。我们发现，胚胎在产卵后的头六到八小时内，随着年龄的增长，其通透性会下降。卵黄膜的这种明显的发育变化导致了即使在接近发育阶段的胚胎中也能看到通透性的异质性。然而，利用罗丹明B染料的荧光特性和不同的EPS处理条件，我们证明了获得最佳渗透性的可存活胚胎的能力。我们还展示了使用早期和晚期发育终点来评估几种化合物在体外应用于胚胎的致畸活性的能力。将该方法应用于携带GFP报告基因的转基因菌株，可以很好地读出胚胎发生的药理变化。制备一批渗透性胚胎所需的直接和快速的操作具有建立果蝇胚胎作为毒理学替代模型和以高通量形式进行小分子筛选的潜力。
Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of D-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995 Daltons. We find that the embryo undergoes an age dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high throughput format.
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DOI：
10.1002/neu.20020
发表时间：
2004-09-05
期刊：
JOURNAL OF NEUROBIOLOGY
影响因子：
--
作者：
Mellerick, DM;Liu, H
通讯作者：
Liu, H
影响因子：
2.6
作者：
PAPASSIDERI, I;MARGARITIS, LH;GULIKKRZYWICKI, T
通讯作者：
GULIKKRZYWICKI, T
影响因子：
4.6
作者：
DeLotto, Robert;DeLotto, Yvonne;Lippincott-Schwartz, Jennifer
通讯作者：
Lippincott-Schwartz, Jennifer
影响因子：
2.4
作者：
Cernilogar, FM;Fabbri, F;Gargiulo, G
通讯作者：
Gargiulo, G
影响因子：
1.6
作者：
STRECKER, TR;MCGHEE, S;HAM, D
通讯作者：
HAM, D