FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors
FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors
复制标题
FasL 诱导 Fas/Apo1 介导的人胚肾 293 细胞凋亡，该细胞通常用于生成 E1 缺失的腺病毒载体
作者：
A. Larregina;Adrian E. Morelli;Ricardo A. Dewey;Maria G. Castro;Adriano Fontana;P. Lowenstein
人胚胎肾293细胞含有5型腺病毒的E1区，因此，通过易体互补，可以产生E1缺失的重组腺病毒载体。在一个强截断的直接早期人巨细胞病毒（MIEhCMV）启动子控制下，我们试图产生表达凋亡诱导分子Fas配体（FasL）的重组腺病毒，我们发现293细胞在与编码小鼠FasL的穿梭质粒初始共转染后不能存活；pJM17，一种含有5型腺病毒基因组的质粒，在E1-E3区域缺失，以不可包装的形式存在。对共转染后大量细胞死亡原因的研究使我们确定293细胞表达FasL受体Fas-Apo1 (CD95)，并对Fas-Apo1与IgM单克隆抗体或FasL交联产生凋亡反应。因此，我们决定在组织特异性和/或诱导启动子元件的控制下生成表达FasL的腺病毒载体。我们的发现可以解释一些小组在使用293细胞产生表达FasL的重组腺病毒载体以及报道的低滴度时遇到的困难。
Human embryonic kidney 293 cells contain the E1 region of adenovirus type 5, and thus sustain, through transcomplementation, the production of recombinant E1-deleted adenovirus vectors. During attempts to produce recombinant adenovirus expressing the apoptosis-inducing molecule Fas ligand (FasL) under the control of a very strong truncated major immediate–early human cytomegalovirus (MIEhCMV) promoter, we discovered that 293 cells were not surviving the initial cotransfection with a shuttle plasmid encoding the mouse FasL; and pJM17, a plasmid containing the genome of adenovirus type 5 with deletions in the E1-E3 regions, in an unpackagable form. Investigation of the reason for massive cell death after cotransfection led us to determine that 293 cells express the FasL receptor, Fas-Apo1 (CD95), and respond with apoptosis to the cross-linking of Fas-Apo1 with either IgM monoclonal antibodies or FasL. Therefore, we decided to generate adenoviral vectors expressing FasL under the control of tissue-specific and/or -inducible promoter elements. Our findings can explain difficulties several groups have had in generating recombinant adenoviral vectors expressing FasL using 293 cells, as well as the lower titres reported.
非人灵长类动物对长期反复肺部暴露于 Ad2/CFTR-2 的体液和细胞免疫反应。
DOI：
--
发表时间：
1996
期刊：
Gene therapy.
影响因子：
--
作者：
Kaplan,JM;StGeorge,JA;Pennington,SE;Keyes,LD;Johnson,RP;Wadsworth,SC;Smith,AE
通讯作者：
Smith,AE
影响因子：
4.2
作者：
Muruve, DA;Nicolson, AG;Libermann, TA
通讯作者：
Libermann, TA