RESUMO
The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5' polycytosine and 3' polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content.
Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Genoma , Animais , Composição de Bases , Sequência de Bases , Primers do DNA/química , Primers do DNA/genética , DNA de Cadeia Simples/metabolismo , Cabras , Guanina/química , Integrases/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Timidina/químicaRESUMO
The enzyme φC31 integrase from Streptomyces phage has been documented as functional in mammalian cells and, therefore, has the potential to be a powerful gene manipulation tool. However, the activity of this enzyme is cell-type dependent. The more active mutant forms of φC31 integrase are required. Therefore, a rapid and effective method should be developed to detect the intracellular activity of φC31 integrase. We devised in this study an integrase-inversion cassette that contains the enhanced green fluorescent protein (EGFP) gene and the reverse complementary DsRed gene, which are flanked by attB and reverse complementary attP. This cassette can be inverted by φC31 integrase, thereby altering the fluorescent protein expression. Thus, φC31 integrase activity can be qualitatively or quantitatively evaluated based on the detected fluorescence. Furthermore, this cassette-based method was applied to several cell types, demonstrating that it is an efficient and reliable tool for measuring φC31 integrase activity in mammalian cells.
Assuntos
Bacteriófagos/enzimologia , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde/análise , Integrases/metabolismo , Proteínas Luminescentes/análise , Streptomyces/virologia , Animais , Linhagem Celular , Ensaios Enzimáticos , Corantes Fluorescentes/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Integrases/genética , Proteínas Luminescentes/genética , TransfecçãoRESUMO
BACKGROUND: Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae family are causative agents of sheep pox and goat pox respectively, which are important contagious diseases and endemic in central and northern Africa, the Middle and Far East, and the Indian sub-continent. Both sheep pox and goat pox can cause wool and hide damage, and reduce the production of mutton and milk, which may result in significant economic losses and threaten the stockbreeding. In this study, three SPPVs and two GTPVs were collected from China in 2009 and 2011. We described the sequence features and phylogenetic analysis of the P32 gene, GPCR gene and RPO30 gene of the SPPVs and GTPVs to reveal their genetic relatedness. RESULTS: Sequence and phylogenetic analysis showed that there was a close relationship among SPPV/GanS/2/2011/China, SPPV/GanS/1/2011/China and SPPV/NingX/2009/China. They were clustered on the same SPPV clade. GTPV/HuB/2009/China and GS-V1 belonged to the GTPV lineage. GS-V1 was closely related to other GTPV vaccine strains. GTPV/HuB/2009/China and GS-V1 were clustered with GTPVs from China and some southern Asian countries. CONCLUSION: This study may expand the datum for spread trend research of Chinese SPPVs and GTPVs, meanwhile provide theoretical references to improve the preventive and control strategy.