转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港
Summary
TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.
Abstract
Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.
Introduction
重新编程人类体细胞分化成胚胎干细胞样诱导多能干细胞（iPS细胞）的能力，首次发现了高桥等人，于2007年1。转导的逆转录病毒表达4转录因子人真皮成纤维细胞（将这样配成山因子的Oct3 / 4，SOX2，c-Myc的和Klf4）被证明是高度相似基于形态学，增殖，基因表达，和表观遗传状态的人类胚胎干细胞（胚胎干细胞）;关键的是，iPS细胞也能分化成所有三种胚层1的细胞。 iPS细胞的增殖潜力和分化的能力，使他们非常有吸引力的工具;通过重新编程从患有特定疾病的患者的细胞，iPS细胞可被用来作为体外疾病模型系统和作为潜在的治疗剂。
对于后者的目的，有几个问题必须的iPSCs的全部潜力之前得到解决在临床环境，可以实现; 在体外培养的人类胚胎干细胞和iPS细胞，重新编程和细胞维持期间使用异种衍生物的致瘤潜力，并且需要在体内跟踪移植细胞都是至关重要的障碍的临床应用的多能干细胞的（来自Hentze 等人 。 2）。一个理想的解决方案的需要跟踪分化的细胞移植后会涉及目视可检测的标记物抵抗沉默和花斑不管应用程序的。综合转基因稳健和持续的表达是最容易实现的，当外源DNA导入安全港位点;即，基因组位点，使综合向量的足够的转录，而在同一时间减轻表达扰动邻近基因3。一个这样的网站已经非常好特点，因为它的发现是腺相关病毒INTEGRATION站点1（AAVS1），在蛋白磷酸酶1调节亚基12C（PPP1R12C）基因的第一内含子。该位点已被证明不仅以允许持续和通过延长时间鲁棒表达的整合的转基因在培养和体外分化3，但也保护周围的基因从转录扰动4;两个特征被认为是由于内源染色质绝缘体元件侧翼AAVS1部位5的存在。
在刚刚过去的十年进展基因工程工具，极大地促进了易用性和效率与遗传操作的任何细胞类型可以实现的。虽然早期的成功实验，依靠内源性同源重组（HR）的极低水平与引进捐助来实现基因在胚胎干细胞6,7目标，采用特定地点核酸酶，如锌指核酸酶（ZFN），即显通过双链DNA断裂的产生ficantly诱导同源重组大大增加了这样的实验8,9的效率。这两个转录激活因子样效应植物病原黄单胞属（故事）和原核聚集定期相互间隔短回文重复（CRISPR）/ Cas9系统进入高效的站点特定的设计师核酸酶的再利用已经取得了基因打靶的多能干细胞的访问，可行的方法10-13。
最近的一篇论文描述了用于稳定整合绿色荧光报道基因盒的成在使用TALE核酸（TALENS）14人类iPSC的AAVS1安全港轨迹的有效方法。这些有针对性的iPS细胞保持其荧光即使在定向分化为心肌细胞，并移植到心肌梗死（MI）的小鼠模型，提供了强有力的证据，这样的效用稳定地荧光多能干细胞14。以获得目标菌落，基因陷阱方法用于其中一个拼接-受体（SA），2A自我裂解肽序列置于内源性PPP1R12C启动子的控制下的嘌呤霉素N-乙酰基转移酶（PAC）的基因;因此，只有那些掺入该DNA供体的AAVS1轨迹的iPSCs表达PAC，使它们可选择基于嘌呤霉素抗性; （ 图1，15）。该协议的细节产生AAVS1-GFP iPSCs的报道，在最近的一篇文章14，包括iPS细胞转染与TALENS的过程和9.8 kb的捐助为4.2kb DNA片段插入AAVS1安全港轨迹整合，选择iPS细胞的基础上，程序嘌呤霉素抗性，和用于克隆扩增拾取菌落。本文描述的技术可以应用到许多基因组工程实验。
Protocol
Representative Results
Discussion
对于成功的一代AAVS1安全港的最关键的步骤有针对性的人类iPSCs的是：（1）有效地提供TALEN和捐助质粒成iPS细胞转染; （2）优化的iPSC成单个细胞在转染后的转染和铺板密度之前的解离; （3）优化剂量和基于iPSC系的生长的药物选择的时间; （4）认真剖析，并有针对性的挑选殖民地，转移到新的板/孔。相比于Hockemeyer的纸张10用类似的方法，该协议中使用的一对开源AAVS1-TALENS的，不同的iPSC解离…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.
Materials
| NAME OF MATERIAL/EQUIPMENT | COMPANY | CATALOG # | COMMENTS/DESCRIPTION |
| Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL | Corning | 354230 | Store at -20°C. |
| DMEM/F-12 | Life Technologies | 11320-033 | Store at 4°C. |
| Costar 6 Well Clear TC-Treated Multiple Well Plates | Corning | 3506 | |
| Essential 8 Medium | Life Technologies | A1517001 | Store basal medium at 4°C. Store supplement at -20°C. |
| Y-27632 dihydrochloride | Tocris | 1254 | Store at room temp. Once dissolved in H2O, store at -20°C. |
| Sodium Chloride | Sigma | S5886-500G | |
| UltraPure 0.5M EDTA, pH 8.0 | Life Technologies | 15575-020 | |
| DPBS, no calcium, no magnesium | Life Technologies | 14190-250 | |
| 100mm TC-Treated Culture Dish | Corning | 430167 | |
| DR4 MEF 2M IRR – Academic | GlobalStem | GSC-6204G | Store in liquid Nitrogen. |
| DMEM, high glucose, pyruvate | Life Technologies | 11995-040 | Store at 4°C. |
| Defined Fetal Bovine Serum, US Origin | HyClone | SH30070.03 | Store at -20°C. Thaw at 4°C overnight and aliquot. Store aliquots at -20°C until needed. |
| MEM Non-Essential Amino Acids Solution (100X) | Life Technologies | 11140-050 | Store at 4°C. |
| 4D-Nucleofector Core unit | Lonza | AAF-1001B | part of the electroporation system |
| 4D-Nucleofector X unit | Lonza | AAF-1001X | part of the electroporation system |
| P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT) | Lonza | V4XP-3024 | Upon arrival, remove Primary Cell Solution and supplement and store at 4°C. |
| StemPro Accutase Cell Dissociation Reagent | Life Technologies | A1110501 | Store at -20°C. Thaw overnight at 4°C and warm an aliquot in a 37°C water bath before use. |
| NutriStem XF/FF Culture Medium | Stemgent | 01-0005 | Store at -20°C. Thaw overnight at 4°C |
| AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1) | Addgene | 52637 and 52638 | |
| AAVS1-CAG-EGFP Homologous Recombination donor | Addgene | 22212 | |
| Puromycin Dihydrochloride | Life Technologies | A11138-03 | Store at -20°C. Prepare working aliquots of 1 mg/mL in ddH2O. |
| Disposable Borosilicate Glass Pasteur Pipets | Fisher Scientific | 13-678-20A | |
| Sorvall Legend XTR (Refrigerated), 120V 60Hz | Thermo Scientific | 75-004-521 | |
| TX-750 4 × 750mL Swinging Bucket Rotor | Thermo Scientific | 75003607 | |
| Trypan Blue Solution, 0.4% | Life Technologies | 15250-061 |
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