| RRC ID |
69608
|
| 著者 |
Baba Y, Nakano M, Yamada Y, Saito I, Kanegae Y.
|
| タイトル |
Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells.
|
| ジャーナル |
Microbiol Immunol
|
| Abstract |
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.
|
| 巻・号 |
49(6)
|
| ページ |
559-70
|
| 公開日 |
2005-1-1
|
| DOI |
10.1111/j.1348-0421.2005.tb03753.x
|
| PII |
JST.JSTAGE/mandi/49.559
|
| PMID |
15965304
|
| MeSH |
Adenoviridae / genetics*
Cell Line
Cell Proliferation / drug effects
Gene Expression Regulation, Viral
Genetic Vectors / genetics
HeLa Cells
Humans
Integrases / genetics*
Integrases / metabolism
Integrases / pharmacology
Promoter Regions, Genetic
Recombinant Proteins / pharmacology
T-Lymphocytes
Transcriptional Activation
Viral Proteins / genetics
Viral Proteins / metabolism
Viral Proteins / pharmacology*
|
| IF |
1.566
|
| リソース情報 |
| ヒト・動物細胞 |
2-7(RCB1376) |
