爪蟾卵母细胞的cRNA微注射

来自南非爪蛙非洲爪蟾的卵母细胞是经常使用的功能表达系统。

将体外转录的cRNA微注射到卵母细胞的细胞质中，第二天可以测量表达蛋白的功能。 下面简要说明了如何实现这一点。

手术取出卵巢袋（见图）包裹在结缔组织中，该结缔组织中有血管。 有不同成熟阶段的非洲爪蟾卵母细胞。

 使用铂环，将非洲爪蟾卵母细胞机械分离出来。 单个非洲爪蟾卵母细胞的直径为1.0-1.2毫米。 

卵泡细胞层和结缔组织通过胶原酶/高渗EGTA处理去除。 

爪蟾卵母细胞在塑料网格上排列。用约15μm的尖端直径的玻璃毛细管在约5s内将约50nl cRNA的体积液注射入卵母细胞的细胞质中。 

将两个尖端直径分别为1微米的微电极插入非洲爪蟾卵母细胞，并建立一个两电极电压钳位。这样可以测量通过细胞表面膜的离子电流。 

参考文献:

E. Sigel (1987) J. Physiol. (Lond.) 386, 73-90. Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid. 

E. Sigel, R. Baur, G. Trube, H. Möhler and P. Malherbe (1990) Neuron 5, 703-711. The effect of subunit combination of rat brain GABAA receptors on channel function. 

E. Sigel (1990) J. Membrane Biol. 117, 201-221. The use of Xenopus oocytes for the functional expression of plasma membrane proteins. 


制备cRNA注意事项：

RNA transcripts are synthesized from linearized plasmids encoding the desired protein using using the message machine kit (Ambion) according to the recommendation of the manufacturers. A poly(A) tail of about 300 residues is added to the transcripts by using yeast poly(A) polymerase (USB or Amersham). Transcripts are quantified on agarose gels after staining with Radiant Red RNA Stain (Bio-Rad) by comparing staining intensities with various amounts of molecular weight markers (RNA-Ladder, Gibco-BRL). The product is kept at -80°. For expression of multiple subunits, cRNAs coding for these subunits are mixed and stored in water at -80°C.