Torrent C, Berlioz C, Darlix J L
LaboRétro INSERM, Unité de virologie humaine U412, Ecole Normale Supérieure de Lyon, France.
Hum Gene Ther. 1996 Mar 20;7(5):603-12. doi: 10.1089/hum.1996.7.5-603.
The Friend murine leukemia virus (MLV) and VL30 5' leader sequences were recently found to possess an internal ribosomal entry signal (IRES) and promote cap-independent translation of a downstream cistron. The use of an IRES to generate a dicistronic message provides a way to express two exogenous proteins efficiently and stably within cells. In the present study, we used the VL30 and Moloney (Mo)MLV 5' leader sequences with both RNA translation (IRES) and packaging (E/DLS) functions to construct dicistronic retroviral vectors designed to express human placental alkaline phosphatase (plap) and neomycin phosphotransferase (neo). Vectors containing the VL30 (positions 205-794) and MoMLV (positions 210-1,035) 5' sequences between two cistrons were able to direct synthesis of exogenous proteins and were produced at a high titer, indicating that the IRES and packaging elements were functional in such dicistronic retroviral vectors. In addition, long-term selection for neo expression did not impair plap gene activity. In general, no major genetic rearrangement of the integrated recombinant provirus was observed with the dicistronic constructs upon prolonged culture of the infected cells.
最近发现Friend鼠白血病病毒（MLV）和VL30 5'前导序列具有内部核糖体进入信号（IRES），并促进下游顺反子的不依赖帽子的翻译。使用IRES产生双顺反子信息提供了一种在细胞内有效且稳定地表达两种外源蛋白的方法。在本研究中，我们使用具有RNA翻译（IRES）和包装（E/DLS）功能的VL30和莫洛尼（Mo）MLV 5'前导序列构建双顺反子逆转录病毒载体，用于表达人胎盘碱性磷酸酶（plap）和新霉素磷酸转移酶（neo）。在两个顺反子之间包含VL30（第205 - 794位）和MoMLV（第210 - 1035位）5'序列的载体能够指导外源蛋白的合成，并且以高滴度产生，这表明IRES和包装元件在这种双顺反子逆转录病毒载体中具有功能。此外，对neo表达的长期选择并未损害plap基因活性。总体而言，在对感染细胞进行长时间培养后，双顺反子构建体未观察到整合的重组前病毒发生重大基因重排。
