Deciphering protein function during mitosis in PtK cells using RNAi
Deciphering protein function during mitosis in PtK cells using RNAi
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DOI：
10.1186/1471-2121-7-26
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发表时间：
2006-06-23
期刊：
影响因子：
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通讯作者：
Walczak, Claire E.
中科院分区：
文献类型：
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作者：
Stout, Jane R.;Rizk, Rania S.;Walczak, Claire E.
背景：研究有丝分裂需要一个系统，在该系统中，染色体和纺锤体微管的剧烈运动可以可视化。PtK细胞由于其扁平的形态和少量的大染色体，可以很容易地进行显微镜可视化。结果：通过在PtK细胞中进行RNA干扰，我们可以探索许多对纺锤体组装和染色体分离重要的蛋白质的功能。虽然很难将DNA转染到PtK细胞中（效率接近10%），但我们已经以接近100%的效率转染了荧光siRNA。利用cDNA表达文库，我们分离了完整的PtK MCAK（P-MCAK）cDNA。P-MCAK与人MCAK（H-MCAK）蛋白具有81%的同一性，与H-MCAK DNA具有66%的同一性。通过RNAi敲除P-MCAK导致染色体聚集缺陷和纺锤体结构缺陷。活体成像显示，染色体在聚集和分离方面存在缺陷，这与显微注射抑制性抗MCAK抗体后发现的结果相似。因为分离全长克隆是费力的，我们探索使用RT-PCR与简并引物从PtK细胞产生cDNA片段，从其设计siRNA。我们从PtK细胞中分离到一个有丝分裂驱动蛋白Eg 5的cDNA片段。该片段与H-Eg 5蛋白质的同源性为93%，与H-Eg 5 DNA的同源性为87%。在HeLa和PtK细胞中使用保守的21 bp siRNA进行RNAi，其中Eg 5敲低导致有丝分裂指数增加和具有单极纺锤体的细胞。此外，我们使用RT-PCR分离片段的5个额外的基因，其序列的同一性范围从76到90%与人类，小鼠，或大鼠的基因，这表明这种策略是可行的，适用于任何基因的interests.Conclusion：这种方法将使我们能够有效地探测有丝分裂缺陷的蛋白质敲低相结合的基因组信息从其他生物体与易于处理的形态PtK细胞。
Background: Studying mitosis requires a system in which the dramatic movements of chromosomes and spindle microtubules can be visualized. PtK cells, due to their flat morphology and their small number of large chromosomes, allow microscopic visualizations to be readily performed.Results: By performing RNAi in PtK cells, we can explore the function of many proteins important for spindle assembly and chromosome segregation. Although it is difficult to transfect DNA into PtK cells (efficiency similar to 10%), we have transfected a fluorescent siRNA at nearly 100% efficiency. Using a cDNA expression library, we then isolated a complete PtK MCAK (P-MCAK) cDNA. P-MCAK shares 81% identity to Human-MCAK (H-MCAK) protein and 66% identity to H-MCAK DNA. Knockdown of P-MCAK by RNAi caused defects in chromosome congression and defective spindle organization. Live imaging revealed that chromosomes had defects in congression and segregation, similar to what we found after microinjection of inhibitory anti-MCAK antibodies. Because it is laborious to isolate full-length clones, we explored using RT-PCR with degenerate primers to yield cDNA fragments from PtK cells from which to design siRNAs. We isolated a cDNA fragment of the mitotic kinesin Eg5 from PtK cells. This fragment is 93% identical to H-Eg5 protein and 87% identical to H-Eg5 DNA. A conserved 21 bp siRNA was used for RNAi in both HeLa and PtK cells in which Eg5 knockdown resulted in an increased mitotic index and cells with monopolar spindles. In addition, we used RT-PCR to isolate fragments of 5 additional genes, whose sequence identity ranged from 76 to 90% with human, mouse, or rat genes, suggesting that this strategy is feasible to apply to any gene of interest.Conclusion: This approach will allow us to effectively probe mitotic defects from protein knockdowns by combining genomic information from other organisms with the tractable morphology of PtK cells.