Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves v...Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.展开更多
【目的】验证鸡circSFMBT2的环形结构,探究其表达规律及功能。【方法】以麒麟鸡和DF-1细胞为研究对象,根据反向剪切位点序列特征设计引物,通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性,利...【目的】验证鸡circSFMBT2的环形结构,探究其表达规律及功能。【方法】以麒麟鸡和DF-1细胞为研究对象,根据反向剪切位点序列特征设计引物,通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性,利用qRT-PCR探究不同组织、不同时期circSFMBT2的表达水平。通过构建过表达载体,结合qRT-PCR、Edu和CCK-8等试验,探究circSFMBT2对鸡DF-1细胞增殖的影响。【结果】鸡circSFMBT2全长827 nt,由SFMBT2基因外显子12~18环化形成。RNase R耐受性试验表明,circSFMBT2不易被RNase R降解,随机引物对circSFMBT2的反转录效率是Oligo-d(T)18引物的8倍。组织表达谱表明,circSFMBT2在麒麟鸡14胚龄以及1周龄的肝脏和脾脏中高表达、在胸肌和腿肌中低表达。circSFMBT2在胸肌和腿肌的时序表达谱表明,circSFMBT2在胚胎时期表达量较高,出生后表达量迅速下降。在DF-1细胞中circSFMBT2过表达48 h后,增殖标记基因PCNA和CCND1的表达量分别较对照组上调85%和92%。E d u和C C K-8细胞增殖试验表明,鸡c i r c S F M B T 2可以促进细胞增殖进程。【结论】circSFMBT2是鸡SFMBT2基因的一个环状转录本,可以促进DF-1细胞的增殖,研究结果为深入探究鸡circSFMBT2的生物学功能和作用机制奠定了基础。展开更多
文摘Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.
文摘【目的】验证鸡circSFMBT2的环形结构,探究其表达规律及功能。【方法】以麒麟鸡和DF-1细胞为研究对象,根据反向剪切位点序列特征设计引物,通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性,利用qRT-PCR探究不同组织、不同时期circSFMBT2的表达水平。通过构建过表达载体,结合qRT-PCR、Edu和CCK-8等试验,探究circSFMBT2对鸡DF-1细胞增殖的影响。【结果】鸡circSFMBT2全长827 nt,由SFMBT2基因外显子12~18环化形成。RNase R耐受性试验表明,circSFMBT2不易被RNase R降解,随机引物对circSFMBT2的反转录效率是Oligo-d(T)18引物的8倍。组织表达谱表明,circSFMBT2在麒麟鸡14胚龄以及1周龄的肝脏和脾脏中高表达、在胸肌和腿肌中低表达。circSFMBT2在胸肌和腿肌的时序表达谱表明,circSFMBT2在胚胎时期表达量较高,出生后表达量迅速下降。在DF-1细胞中circSFMBT2过表达48 h后,增殖标记基因PCNA和CCND1的表达量分别较对照组上调85%和92%。E d u和C C K-8细胞增殖试验表明,鸡c i r c S F M B T 2可以促进细胞增殖进程。【结论】circSFMBT2是鸡SFMBT2基因的一个环状转录本,可以促进DF-1细胞的增殖,研究结果为深入探究鸡circSFMBT2的生物学功能和作用机制奠定了基础。
