Larsen H J, Brodersen C H, Hjorth J P
Department of Molecular Biology, University of Aarhus, Denmark.
Transgenic Res. 1994 Sep;3(5):311-6. doi: 10.1007/BF01973591.
A 7.1 kb mini-gene construct containing cloned DNA from the murine parotid secretory protein (PSP) gene with 6.2 kb of the promoter, has previously been shown to direct specific mRNA expression to the salivary glands in transgenic mice. However, the level of transgene expression in the parotid gland was only a few percent of the endogenous level. This indicated that elements necessary for high-level expression are still to be found. In this study, we have searched for such regulatory elements in additional flanking regions by using a 25 kb cloned Pspb fragment containing the complete structural gene, 11.4 kb of 5'-flanking sequence, and 2.5 kb 3'-flanking sequence as a transgene. To distinguish the expression of the transgene from that of the endogenous gene, we took advantage of an allelic difference, using an oligonucleotide that recognized the mRNA from Pspb and the transgene but not that from the other allele, Pspa. The expression of the transgene was examined in animals homozygous for Pspa. Three independent integrations all exhibited a level of parotid-gland-specific expression that corresponded to that of the endogenous gene. Thus, sequences responsible for this high-level PSP mRNA expression are situated within the genomic DNA of the transgene.
一个包含来自小鼠腮腺分泌蛋白（PSP）基因的克隆DNA以及6.2 kb启动子的7.1 kb微型基因构建体，先前已被证明能在转基因小鼠中将特定的mRNA表达导向唾液腺。然而，腮腺中转基因的表达水平仅为内源性水平的百分之几。这表明仍需找到高水平表达所需的元件。在本研究中，我们通过使用一个25 kb的克隆Pspb片段作为转基因来寻找额外侧翼区域中的此类调控元件，该片段包含完整的结构基因、11.4 kb的5'侧翼序列和2.5 kb的3'侧翼序列。为了区分转基因和内源基因的表达，我们利用了等位基因差异，使用一种寡核苷酸，它能识别来自Pspb和转基因的mRNA，但不能识别来自另一个等位基因Pspa的mRNA。在Pspa纯合的动物中检测转基因的表达。三个独立的整合都表现出与内源基因相对应的腮腺特异性表达水平。因此，负责这种高水平PSP mRNA表达的序列位于转基因的基因组DNA内。
