Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos.
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.
Nakamura
A
A
Department of Biological Resources and Environmental Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Okumura
J
J
Muramatsu
T
T
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001755:Blastocyst / Q000201:enzymology* / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics*; D006822:Hybrid Cells / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism*; D008845:Microinjections; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics; D019076:Transgenes; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D015678:Viral Structural Proteins; D004247:DNA; D008156:Luciferases