<aside> 💡 Tol2转座机制

Tol2转座子是Koga等人在日本青鳉鱼中发现，属于hAT转座子家族，全长4682bp，末端分别为17bp和19bp的反向重复序列，包含4个外显子，编码蛋白含有649个氨基酸残基。

Tol2作为一个自主的转座元件，自身可以编码转座酶，且迄今为止，是在脊椎动物中发现的唯一具有自主转座活性的转座子。将转座酶编码区序列部分缺失， 但保留转座酶识别和结合区域序列，Tol2便无法发生自主转座。

**优势：**相对更高的转基因效率，无位点特异性，无碱基倾向性

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载体关键元件

• [ ] 5' ITR: Tol2 5' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transposase can recognize them, and insert the flanked region including the two ITRs into the host genome.
• [ ] Promoter: The promoter driving your gene of interest is placed here.
• [ ] Kozak: Kozak consensus sequence. This is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
• [ ] ORF: The open reading frame of your gene of interest is placed here.
• [ ] SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
• [ ] 3' ITR: Tol2 3' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transposase can recognize them, and insert the flanked region including the two ITRs into the host genome.
• [ ] Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
• [ ] pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

• 外源基因的永久整合：

  常规质粒转染只能实现外源基因的瞬时表达，这种外源基因会随着宿主细胞的分裂而不断丢失，在快速分裂的细胞中显得尤为显著。

  Tol2转座子载体和辅助质粒（或者Tol2 mRNA）一起转染到哺乳动物细胞中，由于转座子在转座酶的作用下，目的基因能稳定地整合到宿主细胞的染色体中，从而实现转座子载体上携带的目的基因在宿主细胞中永久表达。

• 技术简单

• **载体容量大：**我们的转座子载体总容量可达11 kb，其中质粒骨架只占3 kb，有足够大的容量可以放置客户所感兴趣的序列。