Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China.
National Clinical Research Center for Child Health of the Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Neuroscience. 2023 Aug 1;524:137-148. doi: 10.1016/j.neuroscience.2023.05.023. Epub 2023 Jun 15.
Vitamin C (VC) is a key antioxidant of the Central Nervous System (CNS) and SLC23A2 (SVCT2) is the only transporter that actively transports VC into the brain. While the existing animal models of VC deficiency are in the whole body, the essential role of VC in brain development remains elusive. In our study presented here, the CRISPR/Cas9 technology was applied for the construction of a C57BL/6J-SLC23A2 em1(flox)Smoc mouse model, which was crossed with the Glial fibrillary acidic protein-driven Cre Recombinase (GFAP-Cre) genotype mice to generate a conditional knockout model of SLC23A2(SVCT2) gene in mice brain (GFAP-Cre;SLC23A2 flox/flox) after generations of crossbreeding. Our results showed that the expression of SVCT2 in GFAP-Cre;SLC23A2 flox/flox (Cre;svct2 f/f) mice brain was significantly decreased, and consistently, the expression of Neuronal nuclei antigen (NeuN), Glial fibrillary acidic protein (GFAP), calbindin-28k, brain-derived neurotrophic factor (BDNF) was down-regulated but Ionized calcium binding adapter molecule 1 (Iba-1) was up-regulated in Cre;svct2 f/f mice brain tissues. On the other hand, the levels of Glutathione, Reduced (GSH), myeloperoxidase (MDA), 8-isoprostane, tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) were significantly increased, but the levels of VC in brain tissue of the model group were decreased in Cre;svct2 f/f mice brain tissues, indicating the protective effect of VC against oxidative stress and inflammation during pregnancy. Thus, the conditional knockout of the SLC23A2 gene in the brain of mouse was successfully established by the CRISPR/Cas9 technology in our study, providing an effective animal model for studying the role of VC in fetal brain development.
维生素 C（VC）是中枢神经系统（CNS）的关键抗氧化剂，SLC23A2（SVCT2）是唯一将 VC 主动转运到大脑的转运体。虽然现有的 VC 缺乏症动物模型是全身性的，但 VC 在大脑发育中的重要作用仍然难以捉摸。在我们目前的研究中，应用 CRISPR/Cas9 技术构建了 C57BL/6J-SLC23A2 em1(flox)Smoc 小鼠模型，该模型与神经胶质纤维酸性蛋白驱动的 Cre 重组酶（GFAP-Cre）基因型小鼠交配，经过几代杂交，在小鼠大脑中产生了 SLC23A2（SVCT2）基因的条件性敲除模型（GFAP-Cre；SLC23A2 flox/flox）。我们的结果表明，GFAP-Cre；SLC23A2 flox/flox（Cre；svct2 f/f）小鼠大脑中的 SVCT2 表达明显降低，相应地，神经元核抗原（NeuN）、神经胶质纤维酸性蛋白（GFAP）、钙结合蛋白-28k（calbindin-28k）、脑源性神经营养因子（BDNF）的表达下调，但离子钙结合接头分子 1（Iba-1）的表达上调。另一方面，模型组小鼠脑组织中的谷胱甘肽、还原型（GSH）、髓过氧化物酶（MDA）、8-异前列腺素、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平显著升高，而 VC 水平降低脑组织中的 VC 水平表明 VC 在妊娠期间对氧化应激和炎症具有保护作用。因此，我们成功地通过 CRISPR/Cas9 技术在小鼠大脑中敲除了 SLC23A2 基因，为研究 VC 在胎儿大脑发育中的作用提供了有效的动物模型。
