Shinoura N, Ohashi M, Yoshida Y, Asai A, Kirino T, Saito I, Hamada H
Department of Molecular Biotherapy Research, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo.
Hum Gene Ther. 1998 Dec 10;9(18):2683-9. doi: 10.1089/hum.1998.9.18-2683.
Generation of a recombinant adenovirus (Adv) that induces the constitutive expression of an apoptotic gene has been extremely difficult owing to severe apoptotic damage to the host cell. In this study, 293 cells were transduced with the caspase-inhibiting CrmA gene (293-CrmA cells), and used as host cells to generate Adv carrying apoptosis-inducing genes (proapoptotic genes). The 293-CrmA cells proved to be highly efficient for the construction of recombinant Adv carrying genes encoding Fas and Fas ligand. Moreover, the 293-CrmA line produced an ample quantity of these recombinant viruses. Because the conventional 293 plaque formation assay did not reflect the actual number of cells infected with the Adv carrying the proapoptotic gene, a determination of the Adv DNA copy number introduced into target cells was necessary to evaluate the quantity of infective virus. The techniques described here should be widely applicable for the construction of a recombinant Adv, in ample quantity, and for the estimation of the quantity of recombinant Adv produced.
由于凋亡基因的组成性表达会对宿主细胞造成严重的凋亡损伤，因此构建能够诱导凋亡基因组成性表达的重组腺病毒（Adv）极其困难。在本研究中，用抑制半胱天冬酶的CrmA基因转导293细胞（293-CrmA细胞），并将其用作宿主细胞来构建携带凋亡诱导基因（促凋亡基因）的Adv。事实证明，293-CrmA细胞对于构建携带编码Fas和Fas配体基因的重组Adv非常高效。此外，293-CrmA细胞系产生了大量的这些重组病毒。由于传统的293噬斑形成试验不能反映感染携带促凋亡基因的Adv的实际细胞数量，因此有必要测定导入靶细胞的Adv DNA拷贝数，以评估感染性病毒的数量。这里描述的技术应该广泛适用于大量重组Adv的构建以及重组Adv产量的估计。
