Identification and Efficient Expression in Ovarian Granulosa Cells of circFDXR in Goat
[Objective]The purpose of this experiment was to identify the expression of circular RNA(circRNA)circFDXR in goat and establish its efficient expression mode in goat primary ovarian granulosa cells.[Method]The cyclic properties of circFDXR were identified by PCR amplification,DNA sequencing,RNase R digestion and Real-time quantitative PCR.Cytoplasmic separation and fluorescence in situ hybridization(FISH)were used to detect the subcellular localization of circFDXR.The linear sequence of circFDXR was seamlessly cloned into the Lentivirus vector pLC5-ciR by homologous recombination,and transfected into 293T cells to package the virus,then the virus titer was determined,and the optimal multiplicity of infection(MOI)was determined.The optimal MOI was used to infect goat primary ovarian granulosa cells.Real-time quantitative PCR was used to detect the overexpression efficiency,and sequencing verified whether circFDXR was cyclized correctly.[Result]Goat circFDXR was mainly localized in the cytoplasm of ovarian granular cells and tolerates digestion of RNase R enzymes.The circFDXR lentiviral overexpression vector was successfully constructed,the concentrated virus titer was 5.49X109 TU/mL,and the optimal MOI for infecting goat ovarian granulosa cells was 100.Real-time quantitative PCR results showed that the expression level of circFDXR in overexpressed group was extremely significantly higher than that in control group(P＜0.01),and the sequencing results showed that the overexpressed circFDXR could be cyclized correctly.[Conclusion]circFDXR was mainly localized in the cytoplasm of granular cells and had higher stability than linear RNA.The circFDXR packaged by Lentivirus could be cyclized correctly in the primary ovarian granulosa cells of goats.The experimental results laid a theoretical basis for further study of the function of circFDXR in the primary ovarian granulosa cells of goats.
万方数据
维普
