Retrovirus vectors designed for efficient transduction of cytotoxic or cytostatic genes
Retrovirus vectors designed for efficient transduction of cytotoxic or cytostatic genes
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设计用于有效转导细胞毒性或细胞抑制基因的逆转录病毒载体
作者：
M. Ui;M. Takada;T. Arai;K. Matsumoto;K. Yamada;T. Nakahata;T. Nishiwaki;Y. Furukawa;T. Tokino;Y. Nakamura;H. Iba
建立稳定的包装细胞系来生产逆转录病毒载体以表达具有细胞毒性或细胞抑制潜力的抗癌基因是很困难的，因为这些基因也会影响包装细胞系的生长。为了克服这个问题，我们设计了一个用于载体RNA生产的转录单元pBabeLPL，其中外源基因的转录被包含嘌呤霉素抗性基因(puro)和poly(A)添加信号的先前插入的存在完全抑制。该插入的两侧是一对串联的 loxP，设计为在引入 Cre 重组酶后被切除，此时外源基因的转录将从 5'-LTR 开始。将携带LacZ或p53作为外源基因的转录单元导入到先前构建的预包装细胞系PtG-S2中，其中VSV-G的表达也被设计为通过引入Cre重组酶来启动，而gag-pol基因则持续表达。通过腺病毒载体引入Cre重组酶后，以高病毒滴度产生了具有设计结构的表达LacZ或p53的VSV-G假型逆转录病毒载体。 p53病毒被证明能够将p53转导到几种人类癌细胞系的整个群体中，并诱导它们在G1期生长停滞，这表明这种载体生产系统将有利于人类基因治疗。
It is difficult to establish stable packaging cell lines producing retrovirus vectors for the expression of anti-oncogenes with cytotoxic or cytostatic potential, because these genes would also affect the growth of the packaging cell lines. To overcome this problem, we designed a transcriptional unit pBabeLPL for vector RNA production, in which the transcription of the exogenous genes is completely suppressed by the presence of a preceding insertion containing the puromycin resistance gene (puro) and a poly(A) addition signal. This insertion is flanked by a tandem pair of loxP, and is designed to be excised after the introduction of Cre recombinase, when transcription of the exogenous gene will be started from the 5′-LTR. The transcriptional unit car- rying LacZ or p53 as the exogenous gene was introduced into a previously constructed prepackaging cell lines PtG-S2, in which the expression of VSV-G is also designed to be initiated by the introduction of Cre recombinase, while the gag-pol gene is expressed continuously. After the introduction of Cre recombinase by an adenovirus vector, LacZ- or p53-expressing VSV-G-pseudotyped retrovirus vectors with the designed structure were produced at high virus titers. The p53 virus was shown to be able to transduce p53 into the entire population of several human cancer cell lines and to induce their growth arrest at the G1 phase, indicating that this vector-producing system will be advantageous for human gene therapy.
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DOI：
10.1093/jnci/86.19.1458
发表时间：
1994-10-05
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
FUJIWARA, T;CAI, DW;ROTH, JA
通讯作者：
ROTH, JA
DOI：
10.1073/pnas.93.19.10057
发表时间：
1996-09-17
影响因子：
11.1
作者：
Chen, ST;Iida, A;Yee, JK
通讯作者：
Yee, JK
DOI：
10.1073/pnas.91.20.9564
发表时间：
1994-09-27
影响因子：
11.1
作者：
YEE, JK;MIYANOHARA, A;FRIEDMANN, T
通讯作者：
FRIEDMANN, T
影响因子：
10.5
作者：
Polyak, K;Waldman, T;Vogelstein, B
通讯作者：
Vogelstein, B
影响因子：
4.2
作者：
Jean Yee Hwa Yang;E. Vanin;M. Whitt;M. Fornerod;R. Zwart;R. Schneiderman;G. Grosveld;A. Nienhuis
通讯作者：
Jean Yee Hwa Yang;E. Vanin;M. Whitt;M. Fornerod;R. Zwart;R. Schneiderman;G. Grosveld;A. Nienhuis