Antisense c-myc effects on preimplantation mouse embryo development.
Antisense c-myc effects on preimplantation mouse embryo development.
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DOI：
10.1073/pnas.89.21.10051
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发表时间：
1992-11
影响因子：
11.1
通讯作者：
B. Paria;S. Dey;G. Andrews
中科院分区：
文献类型：
--
作者：
B. Paria;S. Dey;G. Andrews
反义DNA抑制基因表达是阐明哺乳动物植入前胚胎发育调控机制的一种方法。具体而言，c-myc原癌基因的作用进行了检查。c-myc mRNA和免疫反应性核c-myc蛋白在植入前小鼠胚胎在8细胞/桑椹胚和囊胚阶段的检测表明，这种DNA结合蛋白在早期胚胎发生过程中可能是重要的。研究了c-myc寡脱氧核苷酸（oligos）对小鼠2-细胞胚胎体外发育的影响。在含有与翻译起始密码子互补的未修饰（磷酸二酯）反义c-myc寡核苷酸的培养基中培养的胚胎在8细胞/桑椹胚阶段表现出剂量依赖性的停滞。在较低的浓度（7.5 μ M），这种抑制作用是特异性的反义寡核苷酸，并没有发生与正义链互补或双链体的反义和正义寡核苷酸。然而，在4倍高浓度的DNA（30 μ M）下，所有未修饰的c-myc寡核苷酸都具有胚胎毒性，导致胚胎在2细胞至4细胞阶段停滞。相比之下，几乎所有（98%）与修饰（嵌合硫代磷酸酯/磷酸二酯）反义c-myc寡核苷酸（7.5 μ M）培养的2细胞胚胎在8细胞/桑椹胚阶段表现出发育停滞，而没有发育停滞发生后，与高浓度的修饰的正义补体（30 μ M）孵育。用反义c-myc培养新鲜回收的8细胞胚胎，导致c-myc蛋白的缺失，但在发育囊胚腔的胚胎中表皮生长因子受体没有变化。这些对c-myc的影响是特异性的反义寡核苷酸。这些结果表明，c-myc功能成为特别重要的植入前小鼠胚胎在8细胞/桑椹胚发育阶段，并建立反义DNA可以成功地应用于阐明特定基因在植入前哺乳动物胚胎发育中的作用的方法。
Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mechanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this DNA-binding protein could be important during early embryo-genesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containing an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eight-cell/morula stage. At lower concentrations (7.5 microM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 microM), all unmodified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 microM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with high concentrations of the modified sense complement (30 microM). Culture of freshly recovered eight-cell embryos with antisense c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc function becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development.