构建环状RNA mmu＿circ＿Rab11a质粒载体并转染293T细胞

Construction of circular RNA mmu＿circ＿Rab11a overexpression vector to transfect 293T cells

背景:研究发现Rab11a在细胞自噬、增殖过程中发挥重要作用。由于circ RNA可通过调控亲本基因的表达参与生物学过程的调控,因此,mmu_circ_Rab11a是否同样在细胞自噬、增殖过程中发挥重要作用值得关注。目的:探讨环状RNA mmu_circ_Rab11a质粒载体构建并转染293T细胞的可行性。方法:构建PLC5+mmu_circ_Rab11a过表达质粒载体,采用PCR、基因测序等方法对重组质粒进行验证鉴定。利用脂质体Lipofectamine3000转染试剂将mmu_circ_Rab11a过表达载体瞬时转染至293T细胞中。利用RT-PCR检测对照组、空载组(p LC5-ciR)及mmu_circ_Rab11a过表达组中mmu_circ_Rab11a的基因表达情况并对PCR产物进行Sanger测序验证。mmu_circ_Rab11a过表达质粒载体转染MC3T3,CCK8检测细胞增殖能力。结果与结论:实验成功构建了mmu_Circ_Rab11a过表达载体,可促使293T细胞高效转染mmu_circ_Rab11a。过表达mmu_circ_Rab11a可提高MC3T3细胞的增殖能力。提示可利用基因工程技术构建mmu_circ_Rab11a过表达载体,通过脂质体法转染法转染293T细胞,使其高效转录mmu_circ_Rab11a,为进一步研究mmu_circ_Rab11a的生物功能奠定实验基础。可通过调控mmu_circ_Rab11a的表达影响MC3T3细胞的增殖能力。

BACKGROUND: Rab11a plays an important role in the cell autophagy and proliferation, and circular RNA is involved in the regulation ofbiological processes by regulating the expression of parental genes. Therefore, whether mmu_circ_Rab11a also plays an important role in theprocess of cell autophagy and proliferation deserves further study.OBJECTIVE: To investigate the feasibility of circular RNA (mmu_circ_Rab11a) plasmid vector construction and transfection of 293T cells.METHODS: We constructed PLC5+mmu_circ_Rab11a overexpressed plasmid vectors, verified the recombinant plasmid by PCR and genesequencing. mmu_circ_Rab11a overexpression vector was transiently transfected into 293T cells using Lipofectamine 3000 transfectionreagent. Expression of mmu_circ_Rab11a gene in control, pLC5-ciR and mmu_circ_Rab11a overexpression groups was detected byRT-PCR, and PCR products were verified by Sanger sequencing. mmu_circ_Rab11a overexpression plasmid vector was transfected intoMC3T3 cells and cell counting kit-8 was used to detect cell proliferation ability.RESULTS AND CONCLUSION: mmu_circ_Rab11a overexpression vector was successfully constructed, which could efficiently promotemmu_circ_Rab11a transcribed into 293T cells. Over-expression of mmu_circ_Rab11a could enhance the proliferation of MC3T3 cells. Ourfindings indicate that gene engineering technology can be used to construct mmu_circ_Rab11a overexpression vector, which is thentransfected into 293T cells by liposomal transfection method to make efficient transcription of mmu_circ_Rab11a, laying an experimentalfoundation for further investigation on the biological function of mmu_circ_Rab11a. The proliferation of MC3T3 cells could be affected byregulating the expression of mmu_circ_Rab11a.