| 贠海涛,庞思怡,贺婷,等.LepR 3′UTR缺失导致CreER工具鼠示踪特征改变的研究.骨科,2023,14(1): 68-75. |
| LepR 3′UTR缺失导致CreER工具鼠示踪特征改变的研究 |
| Altered Tracing Characteristics of a CreER Mouse Line due to LepR 3′ UTR Deletion |
| 投稿时间：2022-11-08 |
| DOI：10.3969/j.issn.1674-8573.2023.01.013 |
| 中文关键词: 瘦素受体 关节软骨 骨关节炎 Cre-LoxP系统 |
| 英文关键词: Leptin receptor Articular cartilage Osteoarthritis Cre-LoxP system |
| 基金项目:陕西省创新能力支撑项目计划（2020TD-0036）；陕西省重点研发计划一般项目（2021SF-022）；陕西省重点产业链项目（2022ZDLSF02-12） |
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| 中文摘要: |
| 目的 构建瘦素受体（Leptin receptor，LepR）报告基因（LepR-CreER）小鼠，在体内研究LepR 3′ UTR中引入polyA对骨关节和其他脏器中LepR基因表达模式的影响。方法 通过CRISPR-Cas9基因编辑技术在LepR基因终止密码子前定点敲入2A-CreERT2-WPER-polyA表达框，构建诱导型LepR-CreER基因小鼠，与R26tdTomato小鼠杂交繁育，获得LepR-CreER;R26tdTomato基因小鼠，提取小鼠组织DNA，采用PCR进行基因型鉴定。对幼龄及成年期LepR-CreER;R26tdTomato基因小鼠的骨关节和脏器组织进行冰冻切片，利用DAPI和免疫荧光染色检测体内LepR+细胞表达情况，并与LepR-Cre;R26tdTomato小鼠LepR+细胞表达进行比较。在LepR-Cre和LepR-CreER小鼠关节腔注射Leptin重组蛋白，采用Safranin O染色和Aggrecan免疫荧光染色观察关节软骨的变化。结果 幼龄期，LepR-CreER;R26tdTomato和LepR-Cre;R26tdTomato小鼠的生长板肥大区及骨髓中均有LepR+细胞存在，关节软骨中没有LepR+细胞的出现；而成年期，LepR-CreER;R26tdTomato小鼠关节软骨中LepR+细胞的数目较LepR-Cre;R26tdTomato小鼠明显增多，阳性细胞分布范围扩大。关节腔注射Leptin后，LepR-CreER小鼠较LepR-Cre小鼠出现更严重的关节软骨退变表现。进一步生物信息学及表达对比分析发现LepR基因的3′UTR存在关节软骨中高表达的miR-31-5p互补结合位点。结论 成年期，不同方式构建的LepR报告基因小鼠中LepR+细胞表达具有差异，激活异位表达LepR+细胞的关节软骨出现更严重的骨关节炎，生信分析提示其中的差异可能源自LepR基因3′UTR存在miR-31-5p的潜在结合区域。 |
| 英文摘要: |
| Objective To investigate the impact of polyA konck-in before 3′ UTR on the expression pattern of Leptin Receptor (LepR) in skeleton and internal organs in vivo. Methods We utilized CRISPR-Cas9 gene-editing technology to create LepR-CreER mice by inserting 2A-CreERT2-WPER-polyA signal before the stop codon, and then crossed LepR-CreER mice with R26tdTomato mice to generate inducible LepR-CreER;R26tdTomato mice. The genotype of the LepR-CreER;R26tdTomato mice was verified by polymerase chain reaction (PCR). All limbs were fixed and processed to cryosections. DAPI staining and immunofluorescence staining were used to detect and compare the patterns of LepR+ cells postnatally. Mouse recombinant leptin protein was injected into the joint cavity of the right knee of LepR-Cre and LepR-CreER mice respectively, and the changes of articular cartilage were observed by Safranin O staining and aggrecan immunofluorescence staining. Results At early developmental stages, LepR+ cells were visible within the hypertrophic zone and bone marrow of LepR-CreER;R26tdTomato and LepR-Cre;R26tdTomato mice, but not in articular cartilage. At the adult stage, the number of LepR+ cells in the articular cartilage of LepR-CreER;R26tdTomato mice was significantly increased as compared with that in LepR-Cre;R26tdTomato mice, and the positive range was more extensive. After intra-articular administration of Leptin, LepR-CreER mice displayed more severe cartilage degeneration than LepR-Cre mice. Further bioinformatic analysis revealed that LepR 3′UTR had a specific complementary binding site of miR-31-5p abundantly expressed in articular cartilage. Conclusion The different construction strategies of mouse lines cause differential expression pattern of LepR, and ectopic expression of LepR predisposes the articular cartilage to more severe osteoarthritis. Bioinformatic analysis shows that the existence of binding sites of articular cartilage-expressing miR-31-5p in the LepR 3′UTR might explain the inconsistent articular expression pattern of LepR seen in LepR-Cre;R26tdTomato and LepR-CreER;R26tdTomato mice. |
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