Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells
Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells
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DOI：
10.1111/j.1348-0421.2005.tb03753.x
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发表时间：
2005-01-01
影响因子：
2.6
通讯作者：
Kanegae, Y
中科院分区：
文献类型：
--
作者：
Baba, Y;Nakano, M;Kanegae, Y
位点特异性重组酶 Cre 不仅对于体外而且对于体内基因表达的调节都很有价值。我们之前报道过表达 Cre 的复制缺陷型重组腺病毒 (rAd) 可以在 100% 的培养细胞中介导有效且严格的调控。最近，据报道使用逆转录病毒或慢病毒载体组成型表达 Cre 可抑制细胞生长，但瞬时 Cre 表达的效果尚未得到检验。在这里，我们表明，即使 Cre 瞬时表达，表达 Cre 的 rAd 产生的过量 Cre 也会对细胞产生有害影响。我们使用了三种携带具有不同活性的启动子的rAd：SV40早期启动子(AxSVENCre)、SR α启动子(AxSRCre)和CAG启动子(AxCANCre)。细胞毒性显然是由 Cre 本身引起的，并且当将这些 rAd 的细胞病变效应与缺乏 Cre 表达单元的对照病毒进行比较时，细胞毒性与 rAd 病毒颗粒引起的细胞毒性有区别。 Cre毒性与Cre的表达水平密切相关。重要的是，AxSRCre和AxCANCre给出了60倍的有效MOI范围（“有效范围”），足以激活基因，而不会引起rAd颗粒或Cre本身的细胞毒性，而AxSVENCre未能给出这样的范围，因为Cre的表达水平太低。当Cre被标记核定位信号（NLS）时，不仅其活性而且Cre毒性都增加了四倍，而有效范围没有变化。因此，AxSRNCre 可能比 AxSRCre 更有助于控制 rAd 病毒粒子的细胞毒性。在利用表达 Cre 的 rAd 进行实验之前，通过使用目的细胞系预先检查“有效范围”，可以避免 Cre 诱导的细胞毒性。
The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100 % of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.