Agoston D V, Bravo D T, Waschek J A
Mental Retardation Research Center, Los Angeles, California.
J Neurosci Res. 1990 Dec;27(4):479-86. doi: 10.1002/jnr.490270407.
We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.
我们之前已表明，一种基因构建体，它由5.2 kb的5'侧翼序列、首个外显子以及编码血管活性肠肽（VIP）的人类基因的首个内含子的一部分与报告基因氯霉素乙酰转移酶（CAT）融合而成，当转染到人神经母细胞瘤细胞系SK-N-SH的亚克隆中时，能完全模拟内源性VIP基因的多样行为（Waschek等人，1988年）。为了确定相同的序列是否足以将报告基因的表达靶向小鼠中产生VIP的组织，我们启动了一项初步研究，在该研究中我们生成了四只含有VIP-CAT融合基因的转基因小鼠或小鼠品系。在每个转基因小鼠品系的奠基者或后代的回肠中都检测到了可检测水平的CAT。在所有其他测试组织中，CAT活性要么低于检测水平，要么转基因未表达，但有一只小鼠除外，在其小脑中有异位表达。结果表明，所利用的VIP序列足以将转基因的表达导向肠道，但不一定导向VIP表达的其他位点。为了研究哪些特定的DNA序列可能赋予肠道和其他位点VIP表达，我们使用VIP/荧光素酶融合基因构建体进一步分析了SK-N-SH亚克隆中的VIP基因。发现在VIP转录起始位点上游4.0 kb至4.6 kb之间的一个0.6 kb DNA片段在一个亚克隆中赋予了高水平的表达，并在另一个亚克隆中增强了佛波酯诱导程度。这些以及其他数据表明，多个转录元件控制神经母细胞瘤细胞中VIP的表达，并且是完整动物中VIP基因表达的介导因子候选物。
