青鳉foxl2基因敲除突变体的构建与表型分析

Generation and Phenotype Analysis on foxl2 Mutant in Medaka, Oryzias latipes

【目的】建立青鳉(Oryziaslatipes)foxl2基因突变体材料,为阐释foxl2基因功能提供良好的动物模型和研究基础。【方法】利用CRISPR/Cas9技术敲除青鳉foxl2基因,建立突变体材料。观察foxl2基因突变体的第二性征、性腺等形态特征;组织学分析性腺结构与性腺中细胞类型;用定量PCR检测性腺发育相关基因的表达情况。【结果】通过CRISPR/Cas9技术成功敲除青鳉foxl2基因,建立foxl2基因缺失4个碱基的青鳉突变体。从第二性征与性腺表型上,foxl2-/-突变体均表现为生理雄性;组织切片分析发现,foxl2-/-突变体的性腺结构与野生型卵巢和野生型精巢均有显著差异,存在退化的核仁外周卵母细胞与精子,提示foxl2-/-的性腺在组织结构上类似精巢;定量PCR结果显示,foxl2-/-突变体中雄性性腺发育关键基因sox9b、gsdf、amh等的表达量升高,雌性性腺发育关键基因cyp19a1a等的表达量下降。【结论】通过CRISPR/Cas9技术成功建立青鳉foxl2基因缺失的突变体,foxl2-/-突变体有遗传雌性、生理雄性的表型特征,foxl2对维持青鳉性腺功能有重要作用。

【Objective】Disruption of foxl2 gene in medaka, Oryzias latipes, was generated using CRISPR/Cas9. It provided a new animal model for investigating the function of foxl2 gene in gonadal development. 【Methods】Using CRISPR/Cas9 technique to knock out foxl2 gene, mutant material was generated in Medaka. The morphological characteristics of the mutant material such as secondary sexual characteristics and gonads were observed. Histological analysis was carried out on gonad tissue sections to observe gonad structure, germ cells and somatic cell types of the mutant materials. To comprensively analyze the effects of foxl2 gene on gonadal function, the changes of gene expression level on gonadal development related genes in mutants were detected using qPCR.【Results】The foxl2 gene was successfully disrupted by CRISPR/Cas9 technology, and foxl2 mutants had been generated in this study.According to the phenotype, foxl2-/-mutant is male. The tissue analysis showed that the gonads of foxl2-/-mutant were different with both ovaries of the wild type females and testis of the wild type males. There were degenerated perinucleolar oocytes and spermatids in gonad of mutants. Quantitative real time PCR analysis of the expression of gonadal differentiation and developmental related genes revealed that the loss of foxl2 gene resulted in upregulating the expression of sox9 b, gsdf, and amh, in contrast,downregulating the expression of cyp19 a1 a. 【Conclusion】Disruption of foxl2 gene has been generated in Medaka. Our results revealed the characteristics of foxl2-/-mutants is male in physiology, but female in hereditary. It indicates that foxl2 gene is essential for maintaining gonadal function in Medaka.