Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France.
PLoS One. 2013 Aug 1;8(8):e71032. doi: 10.1371/journal.pone.0071032. Print 2013.
When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.
当犬腺病毒 2 型 (CAdV-2，也常称为 CAV-2) 载体被注射到脑实质中时，它们优先转导神经元，能够有效地向传入区域进行轴突运输，并允许转基因表达至少>1 年。然而，将这些数据转化为用户友好的载体平台一直受到限制，因为 CAV-2 载体的生成具有挑战性。E1 缺失型腺病毒载体的产生通常需要将包含载体基因组的>30kb 的线性 DNA 片段转染到 E1 互补细胞系中。与人类腺病毒 5 型载体的生成相比，CAV-2 载体的生成效率较低，部分原因是复制起始能力降低以及犬科细胞对大的线性 DNA 片段的转染效率差。为了提高 CAV-2 载体的生成效率，我们生成了一种 E1 互补细胞系，该细胞系表达与 I-SceI 融合的雌激素受体 (ER)，I-SceI 是一种酵母 meganuclease，以及包含 I-SceI 识别位点的质粒，这些识别位点侧翼是 CAV-2 载体基因组。通过转染超螺旋质粒和通过 4-OH-他莫昔芬诱导的 I-SceI 核易位释放细胞内基因组，我们将 CAV-2 载体滴度提高了 1000 倍，从而提高了 CAV-2 载体的生成效率。
