Gardner D P, Byrne G W, Ruddle F H, Kappen C
Samuel C. Johnson Medical Research Center, Mayo Clinic, Scottsdale, AZ 85259, USA.
Transgenic Res. 1996 Jan;5(1):37-48. doi: 10.1007/BF01979920.
The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity. Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.
转基因小鼠系统是研究基因功能的强大工具。然而，当分析涉及对正常发育过程至关重要的基因时，转基因小鼠系统的实用性就受到了限制（综述见Hanahan，1989；Westphal和Gruss，1989；Byrne等人，1991）。这是因为在异位或高水平表达的情况下，转基因可能会干扰发育。因此，建立表达这类基因的永久性转基因小鼠品系已被证明是困难的。为了克服这些困难，已经建立了一种二元转基因小鼠系统，称为多重系统（Byrne和Ruddle，1989）。这是一种两级基因激活系统，其中感兴趣基因的表达仅发生在携带编码两种成分的转基因的后代中：反式激活因子和反式应答元件。反式激活因子品系包含编码单纯疱疹病毒VP16蛋白的基因。反式应答元件品系含有与IE启动子相连的感兴趣基因，该启动子包括VP16反式激活因子的识别序列。以前，已经证明在同时携带反式激活因子和反式应答元件转基因的新生后代中，氯霉素乙酰转移酶报告基因具有诱导性（Byrne和Ruddle，1989）。此外，已经证明VP16蛋白的表达对发育无害，并且反式激活似乎具有组织特异性。在这里，使用大肠杆菌β-半乳糖苷酶基因作为反式应答小鼠品系中的报告基因，研究了该系统在小鼠早期胚胎发育中转基因表达的潜力。为了指导VP16的表达，使用了已知在早期发育过程中具有活性的小鼠Hoxc-8启动子。在不同发育阶段分离反式激活因子与反式应答元件杂交产生的胚胎，并对其进行β-半乳糖苷酶活性染色。早在发育的第八天就可以检测到由强烈的β-半乳糖苷酶染色所证明的反式激活。在所有检查的阶段，lacZ反式应答元件基因的表达模式准确反映了控制VP16表达的Hoxc-8启动子的活性。结果表明，多重系统可用于在小鼠胚胎发育早期以空间和时间定义的方式在多种细胞类型中表达反式应答元件转基因。
