China Animal Husbandry and Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (6): 2330-2341.doi: 10.16431/j.cnki.1671-7236.2024.06.008

• Biotechnology • Previous Articles    

Identification and Efficient Expression in Ovarian Granulosa Cells of circFDXR in Goat

LIU Jie1, LI Zhihan1, GUO Conghui1, FENG Guanghang1, XUE Wenzhe1, LIU Dewu1,2,3, LIU Guangbin1,2,3, SUN Baoli1,2,3, GUO Yongqing1,2,3, DENG Ming1,2,3, ZOU Xian4, LI Yaokun1,2,3   

  1. 1. College of Animal Science, South China Agricultural University, Guangzhou 510642, China;
    2. National Engineering Research Center for Livestock and Poultry Breeding, South China Agricultural University, Guangzhou 510642, China;
    3. Guangdong Key Laboratory of Agricultural Animal Genomics and Molecular Breeding, South China Agricultural University, Guangzhou 510642, China;
    4. Guangdong Key Laboratory of Animal Breeding and Nutrition, State Key Laboratory of Livestock and Poultry Breeding, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
  • Received:2023-10-24 Published:2024-06-01

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Abstract: 【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 5.49×109 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.

Key words: goats; circFDXR; lentiviral packaging; ovarian granulosa

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