BioVector® pMX-IRES-GFP 逆转录病毒荧光表达质粒 / BioVector® pMX-IRES-GFP Retroviral Expression Plasmid

通用定义 / General Definition:BioVector® pMX-IRES-GFP 是一种基于 Moloney 鼠白血病病毒 (MoMLV) 构建的高效率逆转录病毒载体。该载体旨在哺乳动物细胞中实现多基因共表达：目标基因（GOI）通过 5' LTR 启动子驱动表达，随后通过 IRES (内部核糖体进入位点) 序列以双顺反子的形式同时表达 GFP (绿色荧光蛋白)。这种设计允许研究人员通过观察绿色荧光或进行流式分选（FACS）来监测病毒转导效率及阳性细胞克隆，广泛应用于造血干细胞、原代 T 细胞转导以及诱导多能干细胞（iPSC）的重编程研究。

BioVector® pMX-IRES-GFP is a high-efficiency retroviral vector based on the Moloney Murine Leukemia Virus (MoMLV). This vector is designed for the co-expression of multiple genes in mammalian cells: the Gene of Interest (GOI) is driven by the 5' LTR promoter, followed by an IRES (Internal Ribosome Entry Site) sequence that allows for the simultaneous, cap-independent translation of GFP (Green Fluorescent Protein) in a bicistronic message. This architecture enables researchers to monitor transduction efficiency and select positive clones via fluorescence microscopy or flow cytometry (FACS). It is widely used in hematopoietic stem cell research, primary T-cell transduction, and iPSC reprogramming.

BioVector® pMX-IRES-GFP 技术说明书 (Technical Datasheet)

中文版说明书 (Chinese Datasheet)

1. 质粒基本信息

• 载体类型： 逆转录病毒载体 (Retroviral Vector)

• 启动子： 5' LTR (Long Terminal Repeat)

• 报告基因： GFP (Green Fluorescent Protein)

• 调控元件： IRES (Internal Ribosome Entry Site)

• 克隆位点： 多克隆位点 (MCS) 位于 5' LTR 与 IRES 之间

• 细菌抗性： 氨苄青霉素 (Ampicillin, 100 mg/L)

• 复制子： pUC ori

2. 核心生物学功能

• 稳定整合： 病毒基因组可随机整合至宿主细胞染色体中，实现长期稳定的基因表达。

• 双顺反子表达： 目标蛋白与 GFP 独立折叠，避免了直接融合可能导致的蛋白功能障碍。

• 高效转导： 特别适用于分裂活跃的细胞（如淋巴细胞、成纤维细胞）。

3. 实验应用方案

• 病毒包装： 需配合包装细胞系（如 Plat-E 或 Plat-A，或 293T 配合辅助质粒 pGag-Pol 和 pVSV-G）。

• 干细胞研究： 用于导入转录因子（如 Oct4, Sox2, Klf4）进行细胞重编程。

• 筛选与鉴定： 转导后 48-72 小时通过流式细胞术 (FACS) 富集 GFP 阳性细胞。

4. 注意事项

• 插入片段大小： 建议插入片段小于 4 kb，以保证病毒包装效率和滴度。

• 生物安全： 虽然质粒本身不具传染性，但包装出的病毒颗粒应在生物安全二级 (BSL-2) 环境下操作。

English Datasheet

1. Basic Plasmid Information

• Vector Type: Retroviral Vector

• Promoter: 5' LTR (Long Terminal Repeat)

• Reporter Gene: GFP (Green Fluorescent Protein)

• Regulatory Element: IRES (Internal Ribosome Entry Site)

• Cloning Sites: Multiple Cloning Site (MCS) located upstream of the IRES

• Bacterial Selection: Ampicillin (100 mg/L)

• Replicon: pUC ori

2. Key Biological Functions

• Stable Integration: Allows for the permanent integration of the transgene into the host cell genome for long-term expression.

• Bicistronic Expression: The IRES element ensures that the GOI and GFP are translated as separate proteins from a single mRNA, preserving their native functions.

• Broad Tropism: Highly effective for transducing a variety of dividing mammalian cell types, including primary immune cells.

3. Experimental Applications

• Viral Packaging: Requires specialized packaging lines (e.g., Plat-E or Plat-A) or transient transfection of 293T cells with Gag-Pol and Envelope (e.g., VSV-G) plasmids.

• iPSC Reprogramming: One of the gold-standard vectors for delivering Yamanaka factors to somatic cells.

• Efficiency Monitoring: Visualizing GFP expression provides a direct read-out of successful viral infection and integration.

4. Key Usage Notes

• Titer Optimization: Viral titers can be significantly affected by the size of the GOI; keep the total viral genome size within the packaging limit of MoMLV (~7-8 kb).

• Target Cell Proliferation: Retroviruses derived from pMX require host cell division for successful nuclear entry and integration.

注意 / Note: 质粒应储存在 -20 摄氏度。在使用 IRES 载体时，目标基因的表达水平通常高于 IRES 后的报告基因 (GFP)，在分析定量结果时需考虑此特性。Store at -20°C. Note that in IRES-based systems, the expression level of the upstream gene (GOI) is typically higher than that of the downstream reporter (GFP); account for this differential when quantifying expression.

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