利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的研究

Rapid Verification of the Gene Expression in Eukaryotic Plasmid Using Zebrafish Embryo

目的建立利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的实验体系。方法选20枚斑马鱼受精卵,在显微镜下每隔1h记录胚胎的发育情况。另选250枚单细胞期斑马鱼胚胎,平均分成5组,一组胚胎作为对照,剩余4组分别向胚胎的单细胞内注射pEGFP-N1(真核表达质粒)、pCMV-DsRed-Express2(真核表达质粒)、pET28-GFP(原核表达质粒)、pET28-RFP(原核表达质粒)质粒,在不同时间点连续观察绿色荧光及红色荧光的表达情况。另选600枚单细胞期斑马鱼胚胎,平均分成3组,一组胚胎作为对照,一组向胚胎单细胞内注射pEGFP-N1质粒,另外一组向胚胎单细胞内注射pEGFP-N1-MUC1外源基因融合重组质粒,注射4h后在荧光显微镜下观察绿色荧光的表达情况,并用RT-PCR的方法检测目的基因MUC1mRNA的转录情况。结果注射pEGFP-N1、pCMV-DsRed-Express2真核表达质粒的胚胎,注射4h后分别观察到很强的绿色荧光及红色荧光;注射pET28-GFP、pET28-RFP原核表达质粒的胚胎,10h内都未观察到绿色荧光及红色荧光;注射pEGFP-N1-MUC1外源基因融合质粒,注射4h后同样观察到很强的绿色荧光,且用RT-PCR方法检测到目的基因MUC1很强的表达。结论利用斑马鱼胚胎鉴定真核重组质粒中目的基因的表达情况是一种快速、有效的实验体系。

Objective Development of an experimental system to rapidly verify the gene expression of eukaryotic plasmid in zebrafish embryos.Methods 20 zebrafish embryos were selected to observe the developmental stages of the embryo every 1 h.Another 250 one-cell stage embryos were divided randomly into 5 groups,which were injected with pEGFP-N1（eukaryotic expression vector）,pCMV-DsRed-Express2（eukaryotic expression plasmid）,pET28-GFP（prokaryotic expression plasmid）,pET28-RFP（prokaryotic expression vector） and ddH2O,the injected embryos were observed at different time points under fluorescence microscope.Another 600 one-cell stage embryos were divided randomly into 3 groups,which were injected with pEGFP-N1,pEGFP-N1-MUC1（co-expression GFP and MUC1） and ddH2O,the injected embryos were observed at 4 h after injection and RT-PCR was used to detect the mRNA expression of MUC1.Results Strong green and red fluorescence were observed in zebrafish embryos injected with pEGFP-N1,pCMV-DsRed-Express2 plasmids,respectively at 4 h after injection.However,no green or red fluorescence was observed in embryos injected with pET28-GFP or pET28-RFP even in 10 h after injection.In addition,strong green fluorescence was also observed in embryos injected with pEGFP-N1-MUC1 recombinant plasmid at 4 h after injection,and the mRNA of MUC1 was detected by RT-PCR.Conclusion Zebrafish embryo is an effective experimental system to rapidly verify the gene expression of eukaryotic plasmids.