Hantzopoulos P A, Sullenger B A, Ungers G, Gilboa E
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Proc Natl Acad Sci U S A. 1989 May;86(10):3519-23. doi: 10.1073/pnas.86.10.3519.
This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.
本研究描述了一种称为双拷贝（DC）载体的逆转录病毒载体，其设计目的是提高转导基因的表达。DC载体的独特之处在于，转导基因插入到3'长末端重复序列（LTR）的U3区域内。因此，在受感染的细胞中，该基因被复制并转移到5' LTR。重要的结果是，在其新位置，该基因位于逆转录病毒转录单元之外，消除或至少减少了逆转录病毒转录单元的负面影响。通过使用一个2.1千碱基对（kbp）长的腺苷脱氨酶（ADA；EC 3.5.4.4）小基因来测试DC载体设计的实用性，该小基因被插入到N2逆转录病毒载体的3' LTR中，产生了一个2.7 kbp长的嵌合LTR。DNA印迹分析表明，嵌合LTR在感染相应病毒的细胞中被忠实地复制，产生了两个ADA小基因拷贝，每个LTR中各有一个拷贝。与携带相同载体且ADA小基因插入两个LTR之间的细胞相比，将ADA小基因插入N2载体的3' LTR导致NIH 3T3细胞中ADA转录本和人ADA同工酶的合成增加了10至20倍。在测试的两个人类淋巴细胞系HUT 78和Raji中也观察到了类似的ADA表达增加。这些结果与之前的观察结果一致，即上游启动子对下游启动子具有抑制作用，并证实了DC载体设计中所使用的预测。DC载体的使用可能有助于解决在培养细胞中表达逆转录病毒转导基因时遇到的问题，特别是当引入活体动物时。
