一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

Summary

该协议描述了一种方法, 以实现稳定的基因的兴趣进入人类基因组由转录调控的睡美人系统。本文报道了有缺陷的慢载体的合成、人体细胞的体外转导和转细胞的分子检测。

Abstract

睡美人(SB) 转是一种病毒的集成系统, 具有有效的基因转移和功能基因组学的功效。为优化 SB 转机械, 采用转录调节过动转 (SB100X) 和 T2-based 转。通常, 转和转通过质粒转染而瞬时提供, SB100X 表达由本构子驱动。在这里, 我们描述了一个有效的方法, 将 sb 组件传递给那些对几种物理和化学转染方法有抵抗力的人体细胞, 控制 SB100X 表达, 并通过 “剪切和粘贴”, 稳定地整合一个感兴趣的基因 (印度)。机制.自1992年以来, 多动转的表达受到了系统的严格控制, 广泛用于控制基因表达。该基因的兴趣是两侧的反向重复 (IR) 的 T2 转。在 HEK293T 细胞中, 两个部件都被封装在集成缺陷的慢载体上。人类细胞, 无论是细胞系或人类组织的主要细胞, 都是在体外瞬时转与病毒载体。在添加强力霉素 (dox, 四环素模拟) 到培养基中, fine-tuning 的转表达的测量, 并导致长期的基因的兴趣在处理细胞基因组的结合。这种方法是有效的和适用于细胞线 (如, HeLa 细胞) 和原细胞 (如, 人原发角质形成), 从而代表了一个有价值的工具, 基因工程和治疗基因转移。

Introduction

多动 SB100X 转耦合到 T2-based 转已被用于临床前基因治疗应用^1,2,3, 基因组修改^4,5, 以及诱导多能干细胞 (iPSC) 重新编程^6,7。典型地, SB 组分由质粒转染瞬时地提供, 并且一个强的本构子推动转的表示。然而, 尽管改进了新的转染方法, 但两组分系统的交付仍然是许多应用的挑战。因此, 新的交付议定书的发展引起了越来越多的兴趣。除转染方法为质粒⁸和 mRNA⁹, 病毒传递的基础上腺病毒^10,11, 腺相关病毒 (AAV)¹², 病毒性¹³, gammaretroviral¹⁴和慢向量¹⁵已在过去提出。值得注意的是, 选择系统必须保证在不集成病毒载体的情况下暂时交付 SB 部件。然而, 由于转的不可控 rehopping 的风险, 转的本构表达引起了安全问题。

因此, SB100X 的转录调控是第一个挑战。通过将原发育的最小巨细胞病毒 (CMV) 启动子替换为单纯疱疹 Virus-1 (HSV-1)¹⁶的 Timidine 激酶 (TK) 基因的最小启动子 (-81), 在SB100X cDNA (pTetOTKSB100X)。突变的反转四环素-反 rtTA2^s-M2¹⁷是在强本构子 (甘油促进剂, PGK) 的控制下, 在不同质粒 (pCCL-PGKrtTA2^s-M2) 中克隆的。当添加到培养基中时, dox 绑定 rtTA2^s-M2 调制器和系的新的启动子复合体或, 从而导致严格调节的 SB100X 表达式的归纳¹⁸。

第二个主要的挑战是由几种物理和化学方法对人类原细胞的低效转染。为了有效地将转在人类细胞中抗转染, SB100X 和 rtTA2^s-M2 表达式磁带被打包成两个集成缺陷慢向量 (IDLVs)¹⁹: IDLVTKSB 和 IDLVrtTA2^sM2病毒载体瞬时产生作为第三代载体在 HEK293T 细胞²⁰和型的糖蛋白 g 的 Vescicular 口炎病毒 (VSV-g), 它提供了广泛的感染范围。矢量粒子由离心和滴定 HIV-1 塞 p24 免疫。对于传感器细胞系和原细胞, 载体颗粒与凝聚复合, 在 dox 存在或缺失时与靶细胞孵育。用定量 rt-pcr (qRT-pcr)¹⁸测定了转细胞中 SB100X 表达的药物依赖性活化。

一旦春节调控的 SB100X 表达被证明, 将印度 (如绿色荧光蛋白, GFP) 转到靶细胞基因组中的分子事件, 由 Bak 和泰尔杰·密盖尔森²¹清楚地化。在 T2-transposon 国税局 (IDLVT2) 之间复制的一个表达式卡带的第三 IDLV 载体必须在上面提到的构造和包装。最后, 三 IDLV 载体可用于有效地传感器人体细胞 (如, 原发角质形成细胞)在体外, 并在强力霉素¹⁸的存在中整合印度。

Protocol

1. 质粒受雇 注: 质粒 pCCL-PGKrtTA2S-M2 提供了 Prof. Zappavigna (摩德纳大学和雷焦艾米利亚, 摩德纳, 意大利)。pCMVSB100X 质粒携带的多动转和 pT2/BH 转质粒的编码序列由 z 伊维西 (保罗, 兰, 德国) 和 Prof. z Izvak (最大汗分子医学中心, 柏林,德国)。 对于大肠杆菌(大肠杆菌) 中的所有克隆, 请遵循选择和限制酶程序的克隆策略或克隆片段的 PCR 扩增.注:?…

Representative Results

使用这里介绍的程序, 三 IDLV 载体运载被调控的 sb 组分 (SB100X, rtTA2S-M2 和 T2-GFP 转) 被包装并且用于高效率地运载和严密地调控 sb 系统在人的细胞。图 1A显示了一个用于 HeLa 细胞的体外转导的方案, 它是用两个 IDLV 向量的最佳剂量组合 (在表 2中报告) 来评估 SB 转的转录调节。用 qRT PCR (图 1B) 测量了 SB10…

Discussion

在这里, 我们描述一个广泛的方法, 以稳定地整合到目标细胞的基因组通过睡美人介导的转位。尽管 SB 系统的开发是为了提供一种病毒的基因组编辑方法, 但有效地交付集成机械 (转和 T2 转) 是强制性的。因此, 使用 sb 系统在几乎不 transfectable 的主要细胞, 病毒交付 sb 组分在最近几年被追求^10,12,15。然而, 目前使用的病毒?…

Materials

Dulbecco’s modified Eagle’s medium with 4.5g/L glucose w/o L-Glutamine	Lonza	BE12-614F	Cell culture medium.
HyClone Fetal Bovine Serum (U.S.), Characterized	GE Healthcare Life Sciences	SH30071.03	Serum used HEK293T cell culture for lentiviral particle production.
Penicillin 10.000 UI/ml Streptomycin 10.000 UI/ml	Lonza	DE17-602E	Reagents for cell culture medium.
L-Glutamine 200mM	Lonza	BE17-605E	Reagent for cell culture medium.
Sodium Chloride (NaCl)	Sigma-Aldrich	S7653	Reagent for HBS 2X preparation.
HEPES Buffer, 1M Stock in normal saline	Lonza	BE17-737E	Reagent for HBS 2X preparation.
Disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), 99.5%	Merck	106580	Reagent for HBS 2X preparation.
Hydrochloric acid – ACS reagent 37% (HCl)	Sigma-Aldrich	320331	Reagent for HBS 2X preparation.
Sterile water for injection	Fresenius Kabi	–	Reagent for HBS 2X preparation.
0.45 µm PESS filter	Whatman	10462100	Filters used to remove cell debris from viral supernatant.
Polyallomer Beckman tubes	Beckman Coulter	326823	Tubes for concentrating IDLV vectors by ultracentrifugation.
PBS-1X w/o Ca, Mg	Lonza	BE17-516F	Buffer for cell wash and lentiviral particle resuspension.
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A2153	(Optional) Buffer for lentiviral particle resuspension.
HIV-1 Gag p24 immunocapture kit	Perkin Elmer	NEK50001KT	Kit for IDLV particle titration.
Polybrene	Sigma-Aldrich	107689	Reagent to enhance transduction efficiency.
Trypsin (10X) 2.5% in HBSS w/o Ca, Mg	GIBCO	BE17-160E	Reagent to detach HeLa cells (stock solution, to be diluted).
Ethylenediaminetetraacetic acid disodium salt (EDTA)	100938B	AnalaR BDH	Reagent for trypsin working solution preparation.
0.5% Trypsin-EDTA, no phenol red (10X)	GIBCO	15400-054	Reagent to detach keratinocytes (stock solution, to be diluted).
Donor Bovine Serum, New Zealand Origin	GIBCO	16030-074	Serum for Swiss mouse 3T3-J2 cell culture (feeder layer).
Ham’s F12 media	GIBCO	21765	Medium for keratinocytes.
Fetal bovine serum	Lonza	DE14-801F	Serum for HeLa cell culture medium.
Fetal Bovine Serum, qualified, Australia Origin	GIBCO	10099-141	Serum for human primary keratinocyte culture medium.
Insulin from bovine pancreas	Sigma-Aldrich	I5500	Reagents for keratinocyte growth.
Adenine	VWR	1152-25	Reagents for keratinocyte growth.
Hydrocortisone	VWR	3867-1	Reagents for keratinocyte growth.
Cholera Toxin from Vibrio cholerae	Sigma-Aldrich	C8052	Reagents for keratinocyte growth.
Triiodothyronine	Sigma-Aldrich	T5516	Reagents for keratinocyte growth.
Human EGF	Austral Biological	GF-010-9	Reagents for keratinocyte growth.
Mouse monoclonal APC-conjugated Anti-Feeder antibody	Miltenyi Biotech	130-096-099	To label feeder layer.
RNeasy Plus Mini Kit	Qiagen	74134	RNA purification kit.
SuperScript III Reverse Transcriptase	Life Technologies	18080051	Reverse transcriptase kit.
Deoxynucleotide Mix, 10 mM (dNTP)	Sigma-Aldrich	D7295	Reagent for semi-quantitative PCR.
UltraPure DNase/RNase-Free Distilled Water	(any)	–	Reagent for semi-quantitative PCR and for qRT-PCR.
GoTaq G2 Hot Start Polymerase	Promega	M740B	Taq polymerase.
Agarose, for molecular biology	Sigma-Aldrich	A9539	Reagent for agarose gel electrophoresis.
UltraPure TBE Buffer, 10X	Invitrogen	15581028	Reagent for agarose gel electrophoresis, to be dilute (1X) in ddH2O.
Ethidium bromide	Sigma-Aldrich	E1510	Reagent for agarose gel electrophoresis.
Doxycycline	Sigma-Aldrich	D-9891	drug to induce transposase expression in HeLa cells and in keratinocytes
TaqMan Universal PCR Master Mix	Applied Biosystem	4304437	TaqMan Universal PCR Master Mix including primers and probe.
15mL High Clarity polypropylene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile	Falcon Corning	352096	Tubes for cell collection and vector dilution preparation.
150 mm TC-Treated Cell Culture Dish with 20 mm Grid, Sterile	Falcon Corning	353025	Cell culture dish for viral production.
6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid, Individually Wrapped, Sterile	Falcon Corning	353046	Cell culture plate for HeLa and human primary keratinocyte transduction.
Safe-Lock microcentrifuge tubes, volume 1.5 mL	Eppendorf	0030 120.086	Tubes for dilution preparation.
Safe-Lock microcentrifuge tubes, volume 2 mL	Eppendorf	0030 120.094	Tubes for dilution preparation.
0.2ml PCR Tubes with flat caps, RNase, DNase, DNA and PCR Inhibitor free	(any)	–	Tubes for semi-quantitative PCR.
MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL	Applied Biosystem	4346906	96-well for qRT-PCR.
5mL Round Bottom Polystyrene Tube, without Cap	BD Falcon	352052	Tubes for flow cytometry acquisition.
Disposable Serological Pipets, Polystyrene, Individually Wrapped, Sterile (different volumes)	(any)	–	Pipettes for sterile tissue culture applications.
Filter Tips, Sterile, and RNase, DNase, DNA and Pyrogen free (different volumes)	(any)	–	Filter tips for sterile tissue colture and molecular biology applications.
pH meter	(any)	–	Equipment for measurement of HBS 2X pH.
PCR Thermal cycler	(any)	–	Equipment for semi-quantitative PCR.
Agarose gel electrophoresis equipment	(any)	–	Equipment for agarose gel electrophoresis of semi-quantitative PCR.
BD FACS Canto II 2LSR 4/2	BD	–	Flow cytometer.
7900HT Fast Real-Time PCR system	Applied Biosystem	7900HT	Equipment for qRT-PCR.
Benchtop centrifuges for efficient sample processing in cell culture applications, refrigerated, with microplate buckets.	(any)	–	Centrifuge for cell culture processing and spinoculation.
Optima L-90K Ultracentrifuge equipped with SW32TI rotor	Beckman Coulter	–	Ultracentrifuge for lentiviral particle production.
Equipment for cell culture and molecular biology laboratory (e.g., Class II laminar flow hood designed for work involving BSL2 materials, cell culture incubator,refrigerators and freezers (+4 °C, -20 °C, -80 °C), micropipettes and pipet-aid).
Protective laboratory coats, gloves, face protection in accordance with Institute rules and regulations, in particular for lentiviral particle production and transduction.