Masakazu Nakano

Practical Range of Effective Dose for Cre Recombinase-Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells

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 SRα 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.

Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.

Synthesis of antitumor marine steroid aragusterols

Abstract The synthesis of aniilumor marine steroid aragusterols A, B, C and D was successfully carried out. The synthesis involved stereoselectivc construction of side chain segment 8 , coupling of 8 with 20-keto steroid 11 giving aragustcrol B and conversion of aragusterol B into aragusterols A, C and D.

Three new xenicane diterpenoids from Okinawan soft coral of the genus, Xenia

Abstract Three new xenicane diterpenoids, xeniaol, xeniadiol and xeniatriol, were isolated from the Okinawan soft coral of the genus, Xenia . Their structures were elucidated by spectroscopic analysis.

Xeniaoxolane: A New Xenicane-type Diterpenoid from the Okinawan Soft Coral, Xenia sp.; Absolute Configurations of Xeniaoxolane, Xeniolide-A and Xenialactol

Abstract A new xenicane-type diterpenoid, xeniaoxolane, containing tetrahydrofuran, was isolated from the Okinawan soft coral of the genus, Xenia along with xeniolide-A and xenialactol. The relative configuration of xeniaoxolane was determined based on spectroscopic analysis. The absolute configuration of xeniolide-A was determined using the modified Moshers method and those of xeniaoxolane and xenialactol, by chemical conversions.

DNA Substrates Influence the Recombination Efficiency Mediated by FLP Recombinase Expressed in Mammalian Cells

The FLP recombinase derived from Saccharomyces cerevisiae mediates precise site‐specific recombination between a pair of FLP recognition targets (FRTs). Like the Cre/loxP system derived from bacteriophage P1, the FLP/FRT system has recently been applied to gene regulation systems using an FLP‐expressing recombinant adenovirus (rAd) (Nakano et al, Nucleic Acids Res. 29: e40, 2001). In an attempt to improve the FLP/FRT system by altering its DNA substrates, we compared the recombination efficiency among different substrates by a quantitative in vitro assay using FLP expressed in mammalian cells. Unexpectedly, we found that one linearized DNA substrate showed 4‐ to > 20‐fold lower recombination efficiency than other substrates, which phenomenon has not been observed in the Cre/loxP system. The quantitative in vitro assay using truncated DNA substrates suggested that the recombination efficiency seemed to be influenced not only by the linearized position of the substrate, but also by the length between a pair of FRTs. Such substrate preference of FLP expressed in mammalian cells should probably be noted when designing versatile applications of the FLP/FRT system as a gene regulation system in mammalian systems. Fortunately, however, we demonstrated that no substrate preference was observed when using a particular substrate (pCAFNF5) and the preference was reduced when using a certain pair of mutant FRTs (f72), which will also be a promising tool for simultaneous gene regulation in combination with wild‐type FRT.

Generation of neutralizing antibody to the reverse transcriptase of human immunodeficiency virus type 1 by immunizing of mice with an infectious vaccinia virus recombinant

Antibodies inhibiting the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) were found to be generated in the serum of mice repeatedly infected with a vaccinia virus recombinant, WRRT, expressing the enzyme. A monoclonal antibody (mAb), 7C4, which specifically and almost completely inhibits the RNA-dependent DNA polymerase activity of HIV-1 RT was produced from a mouse repeatedly immunized with WRRT. 7C4 seems to be specific for HIV-1 among retroviruses: 7C4 inhibited RT activity of three strains of HIV-1 (IIIB, Bru, and IMS-1) but not of two strains of HIV-2 (GH-1 and LAV-2) or two strains of SIV (MAC and MND). The immunoglobulin isotype of three out of four mAbs produced from spleen cells of the immunized mouse were IgG2a. This immunization method that avoids protein denaturation may preferentially induce a T helper type-1 immune response and increase the chances of producing the only occasionally obtainable mAb capable of recognizing a conformational epitope and completely inhibiting enzyme activity.