Holt J T, Gopal T V, Moulton A D, Nienhuis A W
Proc Natl Acad Sci U S A. 1986 Jul;83(13):4794-8. doi: 10.1073/pnas.83.13.4794.
Antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells by gene transfer techniques. Transcriptional units were constructed consisting of a steroid-inducible mouse mammary tumor virus (MMTV) promoter, mouse or human 5' c-fos gene fragments in either the sense (5' to 3') or antisense (3' to 5') orientation, and splice and poly(A) signals from the human beta-globin gene. A gene that confers neomycin resistance was included in the vectors to allow isolation of stable transformants. Dexamethasone caused a marked induction of hybrid MMTV-fos-globin RNA. Induction of the hybrid transcript containing antisense c-fos RNA decreased colony formation following DNA transfer and inhibited the proliferation of cells into which the antisense transcriptional unit had been integrated. In contrast, colony formation and cell proliferation were not inhibited by induction of hybrid RNA containing c-fos RNA sequences in the sense orientation. These results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes. The c-fos gene product appears to have a required role in normal cell division.
通过基因转移技术在小鼠3T3细胞中产生了与c-fos mRNA互补的反义RNA。构建了转录单位，其由类固醇诱导的小鼠乳腺肿瘤病毒（MMTV）启动子、有义（5'至3'）或反义（3'至5'）方向的小鼠或人类5' c-fos基因片段以及来自人类β-珠蛋白基因的剪接和聚腺苷酸化信号组成。载体中包含赋予新霉素抗性的基因，以允许分离稳定的转化体。地塞米松导致杂种MMTV-fos-珠蛋白RNA的显著诱导。含有反义c-fos RNA的杂种转录本的诱导降低了DNA转移后的集落形成，并抑制了已整合反义转录单位的细胞的增殖。相反，含有有义方向的c-fos RNA序列的杂种RNA的诱导并未抑制集落形成和细胞增殖。这些结果表明，产生反义RNA以抑制基因表达的策略可能有助于阐明原癌基因的功能。c-fos基因产物似乎在正常细胞分裂中具有必需作用。
