BioVector? pMX-IRES-GFP 逆轉錄病毒熒光表達質粒 / BioVector? pMX-IRES-GFP Retroviral Expression Plasmid

通用定義 / General Definition:BioVector? pMX-IRES-GFP 是一種基于 Moloney 鼠白血病病毒 (MoMLV) 構建的高效率逆轉錄病毒載體。該載體旨在哺乳動物細胞中實現(xiàn)多基因共表達：目標基因（GOI）通過 5' LTR 啟動子驅動表達，隨后通過 IRES (內部核糖體進入位點) 序列以雙順反子的形式同時表達 GFP (綠色熒光蛋白)。這種設計允許研究人員通過觀察綠色熒光或進行流式分選（FACS）來監(jiān)測病毒轉導效率及陽性細胞克隆，廣泛應用于造血干細胞、原代 T 細胞轉導以及誘導多能干細胞（iPSC）的重編程研究。

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 技術說明書 (Technical Datasheet)

中文版說明書 (Chinese Datasheet)

1. 質?；拘畔?/strong>

• 載體類型： 逆轉錄病毒載體 (Retroviral Vector)

• 啟動子： 5' LTR (Long Terminal Repeat)

• 報告基因： GFP (Green Fluorescent Protein)

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

• 克隆位點： 多克隆位點 (MCS) 位于 5' LTR 與 IRES 之間

• 細菌抗性： 氨芐青霉素 (Ampicillin, 100 mg/L)

• 復制子： pUC ori

2. 核心生物學功能

• 穩(wěn)定整合： 病毒基因組可隨機整合至宿主細胞染色體中，實現(xiàn)長期穩(wěn)定的基因表達。

• 雙順反子表達： 目標蛋白與 GFP 獨立折疊，避免了直接融合可能導致的蛋白功能障礙。

• 高效轉導： 特別適用于分裂活躍的細胞（如淋巴細胞、成纖維細胞）。

3. 實驗應用方案

• 病毒包裝： 需配合包裝細胞系（如 Plat-E 或 Plat-A，或 293T 配合輔助質粒 pGag-Pol 和 pVSV-G）。

• 干細胞研究： 用于導入轉錄因子（如 Oct4, Sox2, Klf4）進行細胞重編程。

• 篩選與鑒定： 轉導后 48-72 小時通過流式細胞術 (FACS) 富集 GFP 陽性細胞。

4. 注意事項

• 插入片段大小： 建議插入片段小于 4 kb，以保證病毒包裝效率和滴度。

• 生物安全： 雖然質粒本身不具傳染性，但包裝出的病毒顆粒應在生物安全二級 (BSL-2) 環(huán)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: 質粒應儲存在 -20 攝氏度。在使用 IRES 載體時，目標基因的表達水平通常高于 IRES 后的報告基因 (GFP)，在分析定量結果時需考慮此特性。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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