CAJ | 학술논문

通过网上提供的生物信息学分析软件进行搜索和比对,初步筛选到3个较好的针对小鼠双尾-C(Biccl)基因的RNA干扰(RNAi)序列.合成这3个干涉序列片段后克隆到pRS-Hush shRNA载体中.构建Biccl基因的真核表达载体pEGFP-C3-Biccl,将绿色荧光蛋白(GFP)标签标记在Biccl蛋白上.利用细胞转染技术将pEGFP-C3-Biccl与3个干涉序列载体共转染至体外培养的HEK-293细胞中,最后通过细胞荧光强度、半定量PCR和Western blotting鉴定出其中两个序列(pRS-Hush-RNAi-Biccl-N/-C)能明显降低Biccl蛋白在HEK-293细胞中的表达水平,为下一步建立起低表达Biccl的稳定细胞株和研究小鼠Biccl的功能提供了良好的材料.
통과망상제공적생물신식학분석연건진행수색화비대,초보사선도3개교호적침대소서쌍미-C(Biccl)기인적RNA간우(RNAi)서렬.합성저3개간섭서렬편단후극륭도pRS-Hush shRNA재체중.구건Biccl기인적진핵표체재체pEGFP-C3-Biccl,장록색형광단백(GFP)표첨표기재Biccl단백상.이용세포전염기술장pEGFP-C3-Biccl여3개간섭서렬재체공전염지체외배양적HEK-293세포중,최후통과세포형광강도、반정량PCR화Western blotting감정출기중량개서렬(pRS-Hush-RNAi-Biccl-N/-C)능명현강저Biccl단백재HEK-293세포중적표체수평,위하일보건립기저표체Biccl적은정세포주화연구소서Biccl적공능제공료량호적재료.
Based on the online bioinformatics analysis and alignment results, three RNAi sequences target to the Mus musculus Biccl gene were obtained. The three interference fragments were synthesized and cloned into pRS-Hush shRNA Vector. The Biccl eukaryotic expression vector pEGFP-C3-Biccl was constructed, tagging the GFP to the N-terminal of the Biccl protein. The pEGFP-C3-Bicel and three pRS-Hush-RNAi were eo-transfected into the cultured HEK-293 cells line, respectively. The two RNAi (pRS-Hush-RNAi-Biccl-N/-C) that could knock-down the Bicel expression levels in HEK-293cells significantly were confirmed by cell immunofluorescent staining, semi-quantitative PCR and Western blotting. The results demonstrate that we have successfully obtained two efficent Biccl RNAi sequences, which lays a foundation for further studying on the construction of Biccl knock-down stable cell lines and biological function of mouse Biee 1 product.