生命科学前沿讲座课件精品.ppt

* ?Wuhan University of Technology Xie H. RNA interference in mice Approaches for whole organism expression: Express short hairpin RNA (shRNA) in live mice: - Introduce into mouse embryonic stem cells and use standard transgenic techniques to build transgenic mice OR - Inject directly into fertilized eggs and implant in mouse uterus Approaches for tissue-specific expression Inject siRNA directly into targeted organ or tissue OR Use shRNA expression vector driven by tissue-specific promoter to build transgenic mice * ?Wuhan University of Technology Xie H. RNA interference in mice Kunath et al., 2003 Nature Biotech. 21: 559-561 * ?Wuhan University of Technology Xie H. RNA interference in mice * ?Wuhan University of Technology Xie H. RNA interference in mice * ?Wuhan University of Technology Xie H. RNA interference in mice * ?Wuhan University of Technology Xie H. Additional effects of siRNA * ?Wuhan University of Technology Xie H. Additional effects of siRNA siRNA mediated transcriptional silencing siRNA targeted against gene promoters can facilitate DNA methylation and heterochromatin-mediated silencing Previously observed in yeast, flies, and plants Recently described in human cells: Kawasaki Taira 2004 Nature 431: 211-217 * ?Wuhan University of Technology Xie H. FLS 13-5 Guidlines for designing siRNAs * ?Wuhan University of Technology Xie H. Designing siRNAs Guidelines – Test several siRNAs targeting different regions of gene – Thermodynamic considerations in designing siRNAs: High internal stability at the 5’ sense strand Low internal stability at the 5’ antisense strand Absense of internal repeats or palindromes Overall low to medium G+C content (30-50%) – Base preferences Presence of A at positions 3 and 19 of sense strand Absence of G or C at position 19 of sense strand Presence of a U at position 10 of sense strand For more details, see: Mittal 2004 Nat. Rev. Gen. 5: 355-365 – Avoid sequences with strong homology to other genes in the genome * ?Wuhan