BioVector® pT3-EF1a-Bmi1 基于 Sleeping Beauty (SB) 转座子系统的高效转座质粒  

一、 背景与机制说明  

BioVector® pT3-EF1a-Bmi1 质粒 是基于 Sleeping Beauty (SB) 转座子系统 构建的高效转座质粒。该载体利用强效人类延长因子启动子（EF1a）驱动原癌基因 Bmi1 (B lymphoma Mo-MLV insertion region 1) 的高效稳定表达。Bmi1 是 Polycomb 抑制复合物 1 (PRC1) 的核心蛋白组件，通过表观遗传学调控细胞周期阻滞基因（如 p16INK4a / p19ARF）的转录抑制，广泛应用于干细胞干性维持、肿瘤发生（肝癌、血液瘤等）以及睡美人转基因小鼠模型的建立。

二、 质粒元件与特征  

• 载体骨架：pT3-EF1a (Sleeping Beauty 转座载体)

• 转座元素：5' IR/DR 与 3' IR/DR (Sleeping Beauty 转座酶识别与整合序列)

• 启动子：EF1a (人延长因子 1a 启动子)

• 插入片段：Human / Mouse Bmi1 编码区 (CDS)

• 筛选标记：      

  • 大肠杆菌抗性：Ampicillin（氨苄青霉素 100 μg/mL）

  • 哺乳动物筛选：根据骨架构建包含 Puromycin / Neomycin 或纯共转染筛选

  
  

• 宿主菌株：BioVector® DH5α 或 Stbl3

三、 实验应用与水力动力学注射指南  

1. Sleeping Beauty 转座系统共转染

须与编码转座酶的表达质粒（如 BioVector® pCMV-SB100X）按比例（转座质粒 : 转座酶质粒 = 5:1 到 10:1）共转染细胞或共注射小鼠，方可实现基因组宿主序列的精确转座整合。

2. 小鼠水力动力学尾静脉注射（原位肝癌造模）

1. 质粒配制：准备无内毒素质粒，按小鼠体重计算，将 pT3-EF1a-Bmi1 与协同致癌基因质粒（如 pT3-EF1a-c-Met 或 pT3-EF1a-N-Ras）及 pCMV-SB100X 混合于无菌 0.9% 生理盐水中。

2. 快速推注：注射体积相当于小鼠体重的 8%–10%（例如 20g 小鼠注射 1.6–2.0 mL），要求在 5–8 秒 内匀速快速推入尾静脉。

四、 技术指标简表  

BioVector® pT3-EF1a-Bmi1 Plasmid  

1. Background & Function  

BioVector® pT3-EF1a-Bmi1 Plasmid is a high-efficiency transposon vector based on the Sleeping Beauty (SB) transposon system. Driven by the strong human elongation factor-1 alpha (EF1a) promoter, it achieves robust and persistent expression of the Bmi1 oncogene. As a core component of Polycomb Repressive Complex 1 (PRC1), Bmi1 epigenetically silences cell cycle inhibitors (such as p16INK4a/p19ARF). It is widely utilized in studies of stem cell self-renewal, oncogenesis (e.g., HCC, hematological malignancies), and Sleeping Beauty-mediated transgenic mouse models.

2. Vector Architecture & Features  

• Vector Backbone: pT3-EF1a (Sleeping Beauty Transposon Vector)

• Transposon Elements: 5' IR/DR and 3' IR/DR (Recognition & integration sequences for SB transposase)

• Promoter: EF1a (Human elongation factor 1 alpha promoter)

• Insert Gene: Human / Mouse Bmi1 Coding Sequence (CDS)

• Selection Markers:      

  • Bacterial Selection: Ampicillin (100 μg/mL)

  • Mammalian Selection: Optional Puromycin / Neomycin / Blasticidin or co-transfection screening

  
  

• Host Strain: BioVector® DH5α or Stbl3

3. Application & Hydrodynamic Injection Protocol  

1. SB Transposition System

Must be co-transfected or co-injected with a transposase-expressing plasmid (e.g., BioVector® pCMV-SB100X) at a ratio of transposon : transposase = 5:1 to 10:1 for efficient genomic integration.

2. Hydrodynamic Tail Vein Injection (HCC Mouse Model)

1. Plasmid Mix: Prepare endotoxin-free plasmids. Mix pT3-EF1a-Bmi1 with synergistic oncogene plasmids (e.g., pT3-EF1a-c-Met or pT3-EF1a-N-Ras) and pCMV-SB100X in sterile 0.9% saline.

2. High-Pressure Injection: Inject a total volume equivalent to 8%–10% of the mouse body weight (e.g., 1.6–2.0 mL for a 20g mouse) via tail vein within 5–8 seconds.

4. Technical Data Summary  

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