Jones D E, Cui D M, Miller D M
Department of Medicine, University of Alabama at Birmingham, USA.
Oncogene. 1995 Jun 15;10(12):2323-30.
In order to assess the functional contribution of the human c-myc promoter region in the expression of the c-myc gene, transgenic mouse lines containing a bacterial lac Z gene encoding beta-galactosidase under the control of the human c-myc protooncogene promoter were generated. Transgenic mouse embryos heterozygous for the human c-myc Z transgene demonstrate high amounts of beta-galactosidase activity as early as day 11 of embryogenesis by histochemical staining of whole embryos using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) as substrate, localizing specifically to early spinal cord tissue. beta-galactosidase activity can be demonstrated by histochemical staining in brain tissue of day 14 embryos, localizing mainly to the prefrontal cortex region, while relative amounts of beta-galactosidase in spinal cord tissue are reduced. Determination of specific activity of beta-galactosidase using resorufin-beta-galactopyranoside as substrate in homogenates of whole embryos heterozygous for the human c-myc/lac Z transgene demonstrates significantly elevated beta-galactosidase activity over control embryos in day 11 and day 14 embryos. Surprisingly, cell homogenates of brain tissue from adult G1 generation mice heterozygous for the human c-myc/lac Z transgene demonstrate greater than 10-fold higher specific activity of beta-galactosidase over normal control brain tissue. Specific inhibition of the c-myc/lac Z transgene was also demonstrated in developing embryos using mithramycin given at a dose of 150 micrograms kg-1 d-1 intraperitoneal to pregnant females on days 7-13 of gestation. Both histochemical staining of beta-galactosidase and specific activity assays of day 14 embryos demonstrated significantly lower levels of beta-galactosidase than untreated controls. These results are unique since we are able to detect expression of beta-galactosidase in developing embryonic central nervous system tissue along with adult brain tissue of animals carrying the human c-myc Z transgene and we are able to specifically inhibit expression of the transgene using mithramycin administered in utero.
为了评估人类c-myc启动子区域对c-myc基因表达的功能贡献，构建了转基因小鼠品系，其包含在人类c-myc原癌基因启动子控制下编码β-半乳糖苷酶的细菌lac Z基因。携带人类c-myc Z转基因的杂合子转基因小鼠胚胎，早在胚胎发育的第11天，通过使用5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷（X-Gal）作为底物对整个胚胎进行组织化学染色，就显示出大量的β-半乳糖苷酶活性，且特异性定位于早期脊髓组织。在第14天胚胎的脑组织中，通过组织化学染色可显示β-半乳糖苷酶活性，主要定位于前额叶皮质区域，而脊髓组织中β-半乳糖苷酶的相对含量则降低。以刃天青-β-吡喃半乳糖苷作为底物，在携带人类c-myc/lac Z转基因的杂合子整个胚胎匀浆中测定β-半乳糖苷酶的比活性，结果显示在第11天和第14天的胚胎中，β-半乳糖苷酶活性比对照胚胎显著升高。令人惊讶的是，来自成年G1代携带人类c-myc/lac Z转基因的杂合子小鼠脑组织的细胞匀浆显示，其β-半乳糖苷酶的比活性比正常对照脑组织高10倍以上。在妊娠第7至13天，给怀孕雌性小鼠腹腔注射剂量为150微克/千克/天的光神霉素，也证明了在发育中的胚胎中对c-myc/lac Z转基因有特异性抑制作用。对第14天胚胎进行的β-半乳糖苷酶组织化学染色和比活性测定均显示，β-半乳糖苷酶水平明显低于未处理的对照。这些结果很独特，因为我们能够在携带人类c-myc Z转基因动物的发育中的胚胎中枢神经系统组织以及成年脑组织中检测到β-半乳糖苷酶的表达，并且能够通过子宫内给予光神霉素特异性抑制转基因的表达。
