目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6...目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6J小鼠,针对miR-551b外显子1区域,设计导向RNA(guide RNA,gRNA),构建Cas9载体质粒,将体外转录的Cas9 RNA及gRNA显微注射入小鼠的受精卵并体外培养。将培养合格的胚胎移植到代孕小鼠的输卵管中,待小鼠生育后得到F0代小鼠,使用基因测序确定基因敲除情况,与野生型小鼠繁育后,得到F1代杂合小鼠,F1代小鼠经自交繁育获得F2代小鼠,F3代小鼠由F2代纯合小鼠自交获得,采用电泳鉴定小鼠基因型,RT-PCR检测F3代小鼠组织miR-551b的表达。结果:利用CRISPR/Cas9技术构建模型小鼠得到F0代小鼠,通过测序筛选出缺失目标序列的F0代杂合子小鼠。与WT小鼠繁育后,琼脂糖凝胶电泳及测序筛选出F1代杂合小鼠,同样的方法鉴定并获得F2、F3代基因敲除小鼠,获取F3代纯合小鼠的心脏及下腔静脉样本,RT-PCR结果证实F3代纯合小鼠miR551b表达明显低于WT小鼠(P<0.05),成功敲除miR-551b基因。结论:通过CRISPR-Cas9技术成功构建miR⁃155基因敲除小鼠模型并稳定遗传,为进一步研究提供了有利条件。展开更多
The formation mechanism for the body-centered cubic structure of cluster is proposed and its total energy curve is calculated by the method of a Modified Arrangement Channel Quantum Mechanics. The energy is the funct...The formation mechanism for the body-centered cubic structure of cluster is proposed and its total energy curve is calculated by the method of a Modified Arrangement Channel Quantum Mechanics. The energy is the function of separation R between the nuclei at the center and an apex of the body-centered cubic structure. The result of the calculation shows that the curve has a minimal energy . The binding energy of with respect to was calculated to be 0.8857 a.u. This means that the cluster ofmay be formed in the body-centered cubic structure of .展开更多
Two novel clusters [Mn~Ⅲ_3（μ_3-O）（phendox）3]X·13H_2O（X = Cl（1）, Br（2]） have been obtained from the solvothermal reactions of 1,10-phenanthroline-2,9-dicarbaldehyde dioxime（H_2phendox） with MnCl_2...Two novel clusters [Mn~Ⅲ_3（μ_3-O）（phendox）3]X·13H_2O（X = Cl（1）, Br（2]） have been obtained from the solvothermal reactions of 1,10-phenanthroline-2,9-dicarbaldehyde dioxime（H_2phendox） with MnCl_2·4H_2O or anhydrous MnBr_2, and their structures were characterized by elemental analysis, FT-IR, XRD, TGA, MS and single-crystal X-ray diffraction. It crystallizes in trigonal, space group P3_1/c. X-ray analysis reveals that the neighbouring [Mn_3（μ_3-O）（phendox）_3]＋ cores are linked by C–H···Cl hydrogen bonds and form an infinite supramolecular chain along the c-axis. Neighbouring chains are packed with each other by off-set p-p interactions of the aromatic rings on phenox2-. A 3D supramolecular architecture in a honeycomb topology is formed with 1D hexagonal channel in the dimensions of 13？ × 13？ along the c-axis. The gas adsorption studies show that compound 1·13H_2O is stable upon the removal of guest molecules and the desolvated compound absorbed considerable amount of CO_2.展开更多
成簇规则间隔的短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)技术目前广泛应用于生命医学领域的基础研究及临床应用研究。由于载体在CRISPR/Cas9...成簇规则间隔的短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)技术目前广泛应用于生命医学领域的基础研究及临床应用研究。由于载体在CRISPR/Cas9技术中发挥了重要的作用,如何进一步开发和优化载体系统具有重要的意义。传统的载体大多以病毒载体为主,其递送的效率高,但亦存在插入片段的大小有限、免疫反应、致癌、难以大规模生产甚至脱靶等缺陷;而非病毒纳米载体在一定程度上可解决基因编辑过程中由病毒载体所带来的潜在毒性和容量限制等问题,可能具有更广阔的应用前景。本文主要综述了目前用于CRISPR/Cas9系统递送的非病毒纳米载体,探讨了非病毒纳米载体在递送CRISPR/Cas9系统时可能遇到的主要困难,提出了相应的解决方案和策略,以期为基因治疗和药物研发提供新的参考依据。展开更多
规律间隔成簇短回文重复序列及其相关蛋白9(Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术作为一项基因工程领域革新式的技术,为癌症、遗传性疾病及感染...规律间隔成簇短回文重复序列及其相关蛋白9(Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术作为一项基因工程领域革新式的技术,为癌症、遗传性疾病及感染性疾病等多种重大疾病的治疗提供了极大的帮助.但如何在特定细胞和组织中实现时空调控的精准基因编辑,进而避免脱靶效应,依然是该技术在临床转化领域面临的重要挑战.近年来,通过化学分子和反应实现对CRISPR/Cas9活性的调控已经成为提升这项基因编辑技术效率的重要手段之一.本文综合评述了一些最近报道的化学调控CRISPR/Cas9基因编辑的方法,并对其在临床医学领域的应用前景进行了展望.展开更多
基金The project supported by National Natural Science Foundation of China(Grant No.19974027)the Foundation of Sichuan Provincial Education Committee(Grant No.01LB04)
文摘The formation mechanism for the body-centered cubic structure of cluster is proposed and its total energy curve is calculated by the method of a Modified Arrangement Channel Quantum Mechanics. The energy is the function of separation R between the nuclei at the center and an apex of the body-centered cubic structure. The result of the calculation shows that the curve has a minimal energy . The binding energy of with respect to was calculated to be 0.8857 a.u. This means that the cluster ofmay be formed in the body-centered cubic structure of .
基金supported by Guangdong Ocean University with doctor start-up fund(1112263)the Science and Technology Plan Projects of Zhanjiang City(No.2013B01073)
文摘Two novel clusters [Mn~Ⅲ_3（μ_3-O）（phendox）3]X·13H_2O（X = Cl（1）, Br（2]） have been obtained from the solvothermal reactions of 1,10-phenanthroline-2,9-dicarbaldehyde dioxime（H_2phendox） with MnCl_2·4H_2O or anhydrous MnBr_2, and their structures were characterized by elemental analysis, FT-IR, XRD, TGA, MS and single-crystal X-ray diffraction. It crystallizes in trigonal, space group P3_1/c. X-ray analysis reveals that the neighbouring [Mn_3（μ_3-O）（phendox）_3]＋ cores are linked by C–H···Cl hydrogen bonds and form an infinite supramolecular chain along the c-axis. Neighbouring chains are packed with each other by off-set p-p interactions of the aromatic rings on phenox2-. A 3D supramolecular architecture in a honeycomb topology is formed with 1D hexagonal channel in the dimensions of 13？ × 13？ along the c-axis. The gas adsorption studies show that compound 1·13H_2O is stable upon the removal of guest molecules and the desolvated compound absorbed considerable amount of CO_2.
文摘成簇规则间隔的短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)技术目前广泛应用于生命医学领域的基础研究及临床应用研究。由于载体在CRISPR/Cas9技术中发挥了重要的作用,如何进一步开发和优化载体系统具有重要的意义。传统的载体大多以病毒载体为主,其递送的效率高,但亦存在插入片段的大小有限、免疫反应、致癌、难以大规模生产甚至脱靶等缺陷;而非病毒纳米载体在一定程度上可解决基因编辑过程中由病毒载体所带来的潜在毒性和容量限制等问题,可能具有更广阔的应用前景。本文主要综述了目前用于CRISPR/Cas9系统递送的非病毒纳米载体,探讨了非病毒纳米载体在递送CRISPR/Cas9系统时可能遇到的主要困难,提出了相应的解决方案和策略,以期为基因治疗和药物研发提供新的参考依据。
文摘规律间隔成簇短回文重复序列及其相关蛋白9(Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术作为一项基因工程领域革新式的技术,为癌症、遗传性疾病及感染性疾病等多种重大疾病的治疗提供了极大的帮助.但如何在特定细胞和组织中实现时空调控的精准基因编辑,进而避免脱靶效应,依然是该技术在临床转化领域面临的重要挑战.近年来,通过化学分子和反应实现对CRISPR/Cas9活性的调控已经成为提升这项基因编辑技术效率的重要手段之一.本文综合评述了一些最近报道的化学调控CRISPR/Cas9基因编辑的方法,并对其在临床医学领域的应用前景进行了展望.
