Nakamura A, Okumura J, Muramatsu T
Department of Biological Resources and Environmental Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Mol Reprod Dev. 1998 Apr;49(4):368-73. doi: 10.1002/(SICI)1098-2795(199804)49:4<368::AID-MRD3>3.0.CO;2-L.
This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken beta-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos.
本研究旨在通过使用萤火虫荧光素酶报告基因，定量研究具有病毒和杂交增强子及启动子的基因构建体在牛植入前胚胎中的荧光素酶活性。在实验I中，为检测荧光素酶的稳定性，在显微注射后2天，用光度计测量注射了由SV40早期启动子和增强子驱动的荧光素酶基因（SVEluc）的牛胚胎的生物发光强度。结果表明，由于在5至30分钟的测量期间荧光素酶活性恒定，因此可在30分钟内的任何时间分析生物发光。在实验II中，在显微注射后2天或6天，使用光子成像系统分析注射了四种基因构建体（TKEluc、TK6WEluc、SVEluc和Miwluc）的受精卵的荧光素酶表达。实验II的结果表明，由Miw启动子（RSV LTR和鸡β-肌动蛋白启动子）调控的报告基因在牛桑椹胚和囊胚中的表达比其他三种基因构建体更强。在实验III中，研究了SV40增强子融合到Miwluc载体的荧光素酶cDNA下游时的作用。结果表明，SV40增强子进一步激活了牛植入前胚胎中Miw启动子的荧光素酶活性。因此得出结论，在牛植入前胚胎中测试的基因构建体中，Miw启动子与SV40增强子一起将赋予萤火虫荧光素酶报告基因最强的表达。
