Summary

在小鼠心脏rAAV9的制备过表达或敲除基因

Published: December 17, 2016
doi:

Summary

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Abstract

控制通过在鼠模型的心肌递送遗传物质的表达或特定基因的活性使得基因功能的调查。它们在心脏治疗潜力也可确定。有在小鼠心脏体内分子介入有限的方法。重组腺相关病毒(腺相关病毒)系的基因组工程已被利用作为用于体内心脏基因操作的基本工具。这项技术的具体优点包括高效率,高特异性,低基因组整合率,最小 免疫原性,和最小的致病性。这里,构造,封装,和纯化rAAV9矢量的详细过程进行说明。皮下注射rAAV9成新生幼仔导致鲁棒表达或在小鼠心脏感兴趣的基因(多个)的有效敲低,但不是在肝脏和其他组织。使用心脏 – 技术规格获得ÇTNNT2子,在心脏GFP基因的高表达。此外,靶mRNA是在心脏抑制被利用的rAAV9-U6-shRNA的时候。工作rAAV9技术方面的知识可能对心血管有益的调查。

Introduction

控制各种生物系统的特定基因的表达或活性已经成为在基因功能1的研究的有价值的策略。实现这一目标的一个直接的方法是对操纵核苷酸序列,并产生突变等位基因。虽然进行精确的,有针对性的变化到活细胞的基因组中仍 一个耗时耗力的做法,强大的TALEN和CRISPR / Cas9工具的发展开辟基因组编辑2-5的新时代。对于基因操作更常规的实验室方法重点推出遗传物质(DNA的含有编码序列或siRNA的/的shRNA的RNA)进入细胞表达或击倒感兴趣1,6基因(S)的。

在许多情况下,用于基因操作的主要瓶颈是递送DNA,RNA或蛋白的导入细胞。对于体外研究,高效transfecti上系统已经建立了许多培养的细胞系。然而,在特定的小鼠模型中, 体内基因递送是更具挑战性。有一系列的需要,以实现外源试剂的高效细胞摄取被绕过细胞内外的障碍。其他障碍包括快速清除和交付的材料7,8的持续时间短。规避这些问题的一个策略是使用病毒载体的“载体”或“车辆” 用于体内基因递送。病毒的自然进化的传导属性允许感兴趣进入细胞7,9,10基因的有效提供。许多类型的病毒载体,已经开发和实现不同类型的细胞和器官的小鼠体内基因操作灵活。

最常用的病毒系统包括逆转录病毒,慢病毒,腺病毒,和腺相关病毒(AAV) <s达> 11。逆转录病毒是单链RNA病毒,并且可以有丝分裂过程中引入遗传物质,以稳定的方式在宿主细胞基因组中,提供用于在靶细胞和器官12-14转导的基因的终身表达的潜力。但是,许多类型的逆转录病毒只感染分裂的细胞,以及它们在非分裂细胞中的功效是非常低的15。这限制了其效用的基因传递。慢病毒是逆转录病毒科的一属。从其他逆转录病毒不同,慢病毒可以感染两个分割和非分裂细胞,并已被广泛用于基因转移到有丝分裂后和高度分化的细胞16。慢病毒的生命周期也涉及整合载体DNA导入宿主基因组中。因此,慢病毒介导的基因递送使转导的遗传元件16-18的稳定和长持续时间的表达。但是,此功能可能代表一个双-E在使用这些病毒dged剑操纵基因表达,作为载体DNA的整合可能导致插入诱变 在宿主细胞中,并可能导致假象的影响。腺病毒是另一种广泛使用的基因递送系统。不像逆转录病毒和慢病毒,腺病毒是非集成,并且不与宿主细胞8,10,11,19的基因组完整性干扰。此外,腺病毒可以转染DNA引入许多细胞类型和感染是不依赖于活性细胞分裂19。腺病毒的另一个重要特性是易于矢量纯化的,因为病毒载体有能力要复制19,20。然而,这种系统的主要告诫是腺病毒感染可触发靶细胞和器官19强烈的免疫反应,限制在许多调查其使用,特别是在基因治疗研究。

这些不同类型的比较病毒载体的S,重组腺相关病毒(腺相关病毒)似乎是理想的基因递送系统21,22。它具有最小的免疫原性和致病性23,24。此外,腺相关病毒感染广泛的细胞类型,包括分割和非分裂细胞。在大多数情况下,重组腺相关病毒不整合到宿主基因组中;因此,在靶细胞不希望的基因或基因组的改变的风险是低的22。

最近,重组腺相关病毒系统已被成功地用于DNA编码的蛋白质中,miRNA,shRNA的,和CRISPR-gRNAs的体内递送到小鼠心肌23,25-29。这种方法在心血管领域的研究促进了基础研究和基因治疗的研究。这里,详细过程生成rAAV9载体能够有效地过表达或敲除的小鼠心脏目的基因进行了说明。该协议提供了一个简单而有效的方法在小鼠实验模型中操纵心脏基因表达。

Protocol

在由生物安全委员会和波士顿儿童医院的机构动物护理和使用委员会批准的规程进行所有描述的步骤。波士顿儿童医院与监管的光/暗循环和气候控制无病原鼠标设施。兽医和动物关爱员工的变化笼,保证了老鼠的健康。该设施是AAALAC认证,并有活跃的动物福利保障认证(AAALAC认证在1992年2月24日动物福利保障授予数量:A3303-01)。小鼠通过CO 2从压缩气体源输送安乐死。组织样品证实心脏速…

Representative Results

为rAAV9施工rAAV9.cTNT :: GFP或rAAV9.U6 :: shRNA的质粒的策略示于图1和2,分别为。作为例子,在生成rAAV9矢量过表达GFP基因在小鼠心脏。所得质粒含有由两个ITR位点( 图1)侧翼的肌钙蛋白:: GFP盒。所述rAAV9.U6 :: shRNA的载体构建拦截TRBP的mRNA( 图2) 用通过线性回归的qPCR数据生成rAAV9滴…

Discussion

质粒构建中,以尽量减少不希望的ITR重组是重要的。产生病毒之前,必须始终监视用限制性消化和琼脂糖凝胶电泳所述AAV质粒的ITR完整性。这是不可能的,获得100%完好的质粒,但重组比值应该被最小化,尽可能。小于20%是成功rAAV9包装上可接受的。值得注意的是,在与低级振荡速度(180-200转)低的温度(30℃)培养所述细菌可减少ITR重组的机会。

重要的是要确保HEK293细胞?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We thank Dr. Zaffar Haque for careful reading of the manuscript. We thank Drs. Masaharu Kataoka and Gengze Wu for discussions and help. Work in the Wang lab is supported by the American Heart Association, Muscular Dystrophy Association, and NIH (HL085635, HL116919, HL125925).

Materials

Polyethylenimine, Linear (MW 25,000) Polysciences, Inc.  #23966-2
Tube, Polypropylene, 36.2 mL, 25 x 87 mm, (qty. 56) Beckman Coulter, Inc # 362183
Nuclease, ultrapure SIGMA #E8263-25KU
Density Gradient Medium(Iodixanol) SIGMA #D1556-250ML
Centrifugal Filter Unit with Ultracel-100 membrane EMD Millipore Corporation #UFC910008
Laboratory pipetting needle with 90° blunt ends,gauge 14, L 6 in., nickel plated hub SIGMA #CAD7942-12EA
Poloxamer 188 solution (Pluronic® F-68 solution) SIGMA P5556-100ML
Proteinase K SIGMA 3115828001
DNase I Roche 10104159001
Centrifuge machine Thermo Scientific 75004260
Centrifuge System Beckman Coulter 363118
Ultracentrifuge Beckman Coulter
DMEM medium Fisher Scientific SH30243FS
Fetal Bovine Serum  Atlanta Biologicals               S11150
rAAV9 vector Penn Vector Core P1967

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Ding, J., Lin, Z., Jiang, J., Seidman, C. E., Seidman, J. G., Pu, W. T., Wang, D. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (118), e54787, doi:10.3791/54787 (2016).

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