Generation of a conditionally neor-containing retroviral producer cell line: effects of neor on retroviral titer and transgene expression
Generation of a conditionally neor-containing retroviral producer cell line: effects of neor on retroviral titer and transgene expression
复制标题
有条件地含有neor的逆转录病毒生产细胞系的产生：neor对逆转录病毒滴度和转基因表达的影响
作者：
O. Wildner;F. Candotti;E. Krecko;K. Xanthopoulos;W. Ramsey;R. Blaese
我们已经开发了一种方法，用于产生高滴度的逆转录病毒生产细胞系的条件下含有新霉素抗性基因（neor）的基础上的Cre/loxP系统。为此，构建了携带绿色荧光蛋白（GFP）和标记基因盒的双顺反子逆转录病毒剪接载体，该标记基因盒由内部核糖体进入位点（IRES）和侧翼为loxP位点的neor组成，并方便地用于产生G418抗性载体生产细胞系。在滴度测定和验证逆转录病毒上清液的生物活性后，使用腺病毒载体通过瞬时Cre表达从原病毒中切除不再需要的可选择表达盒。该策略导致neor的精确切除和含有功能性“neo-less”逆转录病毒颗粒的逆转录病毒上清液的产生，而对在亲本含有neor的生产系中发现的高载体滴度没有不利影响。GFP表达显着增加后，切除neor，在生产线和逆转录病毒转导的靶细胞。重新引入neor并不改变GFP表达，这表明neor基因和/或其基因产物本身并不充当转录沉默子。
We have developed a method for generating high-titer retroviral producer cell lines conditionally containing a neomycin resistance gene (neor) based on the Cre/loxP system. For this, a bicistronic retroviral splicing vector carrying the green fluorescence protein (GFP) and a marker gene cassette consisting of internal ribosome entry site (IRES) and neor flanked by loxP sites, was constructed and conveniently used to generate a G418 resistant vector producer cell line. Following titer determination and verification of the biological activity of the retroviral supernatants, the selectable expression cassette which was no longer required was excised from the provirus by transient Cre expression using an adenoviral vector. This strategy led to precise excision of neor and generation of retroviral supernatants containing functional ‘neo-less’ retroviral particles without detrimental effects on the high vector titers found in the parental neor-containing producer lines. GFP expression was significantly increased after the excision of neor, in both the producer lines and retrovirally transduced target cells. Reintroduction of neor did not alter GFP expression, suggesting that the neor gene and/or its gene product per se are not acting as a transcriptional silencer.
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DOI：
10.1073/pnas.93.17.8971
发表时间：
1996-08-20
影响因子：
11.1
作者：
Westerman, KA;Leboulch, P
通讯作者：
Leboulch, P
逆转录病毒介导的基因转移至囊性纤维化气道上皮细胞：选择标记序列对长期表达的影响。
影响因子：
14.9
作者：
Olsen,JC;Johnson,LG;Wong-Sun,ML;Moore,KL;Swanstrom,R;Boucher,RC
通讯作者：
Boucher,RC
影响因子：
3.7
作者：
MARKOWITZ, D;GOFF, S;BANK, A
通讯作者：
BANK, A
鼠造血细胞中人腺苷脱氨酶的逆转录病毒基因转移：选择标记序列对长期表达的影响。
影响因子：
20.3
作者：
Apperley,JF;Luskey,BD;Williams,DA
通讯作者：
Williams,DA
影响因子：
3.7
作者：
Li Xu;J. Yee;J. Wolff;T. Friedmann
通讯作者：
Li Xu;J. Yee;J. Wolff;T. Friedmann