Stoeckli Esther T
CSH Protoc. 2006 Sep 1;2006(4):pdb.prot4504. doi: 10.1101/pdb.prot4504.
INTRODUCTIONThis protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi-cell competence changes with differentiation. The half-life of the target protein product may also affect the time point of injection and electroporation-proteins with slow turnover may require RNAi at earlier stages, preferably before the onset of expression. Check the quality of the dsRNA used for injections and make sure that the buffer used for injection does alter development or survival of the embryo. Tris buffers and buffers containing glycerol are not compatible with in vivo injections. Salt concentration and pH should be in the physiological range. Always use the same buffers for injection and electroporation of test and control embryos.
引言
本方案描述了一种用双链RNA（dsRNA）对鸡胚进行胚内转染的方法。将dsRNA注射到胚胎的脊髓中。随后的电穿孔促进了dsRNA分子的细胞摄取。可能需要优化胚胎阶段和电穿孔程序，以提高胚内RNA干扰的有效性——细胞感受态会随着分化而变化。靶蛋白产物的半衰期也可能影响注射和电穿孔的时间点——周转缓慢的蛋白可能需要在更早阶段进行RNA干扰，最好是在表达开始之前。检查用于注射的dsRNA的质量，并确保用于注射的缓冲液不会改变胚胎的发育或存活。Tris缓冲液和含有甘油的缓冲液不适合体内注射。盐浓度和pH值应在生理范围内。对测试胚胎和对照胚胎进行注射和电穿孔时始终使用相同的缓冲液。
