Baba Yasuko, Nakano Masakazu, Yamada Yoshitsugu, Saito Izumu, Kanegae Yumi
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Japan.
Microbiol Immunol. 2005;49(6):559-70. doi: 10.1111/j.1348-0421.2005.tb03753.x.
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.
位点特异性重组酶Cre不仅在体外，而且在体内对基因表达的调控都很有价值。我们之前报道过，表达Cre的复制缺陷型重组腺病毒（rAd）能在100%的培养细胞中介导高效且严格的调控。最近，据报道使用逆转录病毒或慢病毒载体组成型表达Cre会抑制细胞生长，但瞬时表达Cre的效果尚未得到研究。在这里我们表明，即使Cre是瞬时表达的，从表达Cre的rAd产生的过量Cre也会在细胞中造成有害影响。我们使用了三种携带不同活性启动子的rAd：SV40早期启动子（AxSVENCre）、SRα启动子（AxSRCre）和CAG启动子（AxCANCre）。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的效应进行比较时，Cre本身明显导致了细胞毒性，并且与rAd病毒粒子引起的细胞毒性可区分。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倍范围的有效MOI（“有效范围”），足以激活基因而不会因rAd颗粒或Cre本身导致细胞毒性，而AxSVENCre未能给出这样的范围，因为Cre的表达水平太低。当Cre用核定位信号（NLS）标记时，不仅其活性而且Cre毒性增加了四倍，并且有效范围不变。因此，与AxSRCre相比，AxSRNCre可能在控制rAd病毒粒子的细胞毒性方面更有用。在开始利用表达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。
