pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP BioVector®慢病毒基因编辑质粒BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP BioVector® 质粒 / pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP BioVector® Plasmid

数据表 / Datasheet

质粒信息

• 名称：BioVector® pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP 质粒

• 详细名称：含 UCOE 稳定表达元件、SFFV 启动子驱动的 Zim3-dCas9 融合蛋白及 P2A-BFP 报告基因的慢病毒载体

• 载体类型：慢病毒表达载体（第三代 HIV-1 来源）

• 载体骨架：pHR（含 LTR、包装信号、RRE、cPPT/CTS 等元件）

• 宿主菌：推荐使用 BioVector® Stbl3 或 NEB® Stable 感受态细胞，避免重组

• 抗性：氨苄青霉素（Ampicillin，100 μg/mL）

• 拷贝数：低拷贝

• 质粒大小：约 12–14 kb（具体长度以测序为准）

载体元件

培养与保存

• 质粒提取：使用 BioVector® 无内毒素质粒大提试剂盒；建议在含氨苄青霉素的 BioVector® LB 培养基中扩增，30°C 培养以减少重组

• 保存条件：短期 -20°C，长期 -80°C 保存甘油菌或纯化质粒

包装与转导

• 包装细胞：推荐使用 BioVector® HEK293T 细胞

• 包装质粒：需配合第二代或第三代包装辅助质粒（如 BioVector® psPAX2 和 BioVector® pMD2.G）

• 转染试剂：BioVector® 聚乙烯亚胺（PEI）或 BioVector® Lipofectamine™ 3000

• 病毒收获：转染后 48–72 小时收集上清，0.45 μm 滤膜过滤，可浓缩后使用

• 宿主细胞：广泛适用于分裂与非分裂细胞，包括 BioVector® NHEK、BioVector® HEK293、BioVector® Jurkat 等

特征与用途

• CRISPRi 高效抑制：Zim3 是新一代 KRAB 结构域，与 dCas9 融合后可实现对靶基因转录的强效、特异性抑制，脱靶效应低，尤其适合基因功能丧失性研究

• 长期稳定表达：UCOE 元件可抵抗位置效应和启动子沉默，确保在干细胞、原代细胞及体内模型中持续稳定的 CRISPRi 活性

• 报告系统：BFP 荧光报告基因可实时监测病毒转导效率及表达水平，便于分选（流式细胞术）和单克隆筛选

• 单载体系统：所有元件整合于单个慢病毒载体，简化了病毒制备和细胞工程流程

• 应用方向：

  • 基因功能筛选（CRISPRi 文库）

  • 信号通路中关键基因的失活研究

  • 原代细胞（如 T 细胞、神经细胞、角质形成细胞）的基因调控

  • 体内递送及基因治疗模型构建

注意事项

• 该质粒为第三代慢病毒载体，SIN 设计提升了安全性，但仍应在生物安全二级（BSL-2）条件下操作

• 因含有长末端重复序列和重复元件，建议在 30°C 下培养细菌，避免重组缺失

• 使用前应通过测序或酶切验证完整性

Plasmid Information

• Name: BioVector® pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP Plasmid

• Detailed Name: Lentiviral Vector Containing UCOE Stabilizing Element, SFFV Promoter-Driven Zim3-dCas9 Fusion Protein, and P2A-BFP Reporter

• Vector Type: Lentiviral expression vector (third-generation HIV-1 derived)

• Backbone: pHR (containing LTR, packaging signal, RRE, cPPT/CTS, etc.)

• Host Strain: BioVector® Stbl3 or NEB® Stable competent cells recommended to avoid recombination

• Selection: Ampicillin (100 μg/mL)

• Copy Number: Low copy

• Plasmid Size: Approximately 12–14 kb (exact length to be confirmed by sequencing)

Vector Elements

Storage & Handling

• Plasmid Preparation: Use BioVector® endotoxin-free maxi-prep kit; amplify in BioVector® LB medium with ampicillin at 30°C to minimize recombination

• Storage: Short-term at -20°C; long-term at -80°C as glycerol stock or purified plasmid

Packaging & Transduction

• Packaging Cells: BioVector® HEK293T cells recommended

• Packaging Plasmids: Requires second- or third-generation helper plasmids (e.g., BioVector® psPAX2 and BioVector® pMD2.G)

• Transfection Reagent: BioVector® polyethylenimine (PEI) or BioVector® Lipofectamine™ 3000

• Virus Harvest: Collect supernatant 48–72 hours post-transfection; filter through 0.45 μm membrane; may concentrate as needed

• Target Cells: Suitable for dividing and non-dividing cells, including BioVector® NHEK, BioVector® HEK293, BioVector® Jurkat, etc.

Features & Applications

• Efficient CRISPRi: Zim3 is a next-generation KRAB domain; fused with dCas9 it achieves potent and specific transcriptional repression with low off-target effects, ideal for loss-of-function studies

• Long-Term Stable Expression: The UCOE element resists position-effect variegation and promoter silencing, ensuring sustained CRISPRi activity in stem cells, primary cells, and in vivo models

• Reporter System: BFP fluorescent reporter allows real-time monitoring of transduction efficiency and expression; facilitates flow cytometry sorting and clonal selection

• Single-Vector System: All components integrated into one lentiviral vector, simplifying virus production and cell engineering workflows

• Key Applications:

  • Functional gene screening (CRISPRi libraries)

  • Inactivation of key genes in signaling pathways

  • Gene regulation in primary cells (e.g., T cells, neurons, keratinocytes)

  • In vivo delivery and gene therapy model development

Safety & Handling Notes

• This vector is a third-generation lentiviral system with SIN design; standard BSL-2 practices should be followed when handling virus

• Due to the presence of LTRs and repetitive elements, bacterial culture at 30°C is strongly recommended to avoid recombination

• Validate plasmid integrity by restriction digestion or sequencing before use