Department of Ophthalmology, Shunde Hospital of Southern Medical University, Foshan, Guangdong, China.
Department of Ophthalmology, The Fifth Affiliated Hospital of Zunyi Medical University, Zhuhai, China.
Exp Eye Res. 2018 Apr;169:13-19. doi: 10.1016/j.exer.2018.01.017. Epub 2018 Feb 3.
The purpose of this study is to establish a mouse model of transthyretin (TTR) Gly83Arg gene mutation by the technique of gene targeting for research on hereditary vitreous amyloidosis (HVA) and to confirm whether this point mutation is a genetic feature of HVA. A vector (pBR322-MK-TTR) was constructed to target ES cells. The successfully transfected ES cells were used for blastocyst injection, thus generating F0. F0 and Flp mice were mated to generate F1 (TTR, Flp ) mice that lacked the neo gene but carried the Flp gene. F1 mice were mated with C57BL/6N wild type mice to generate F2 (TTR) mice. F3 homozygous and heterozygous mice were generated by mating F2 mice with each other. PCR and sequencing were performed for F3 mice. Amyloid was detected using Congo red stain and polarized light. Immunohistochemistry was used to detect the expression of TTR in the tissues. Quantitative fluorescent PCR and Western blotting were used to detect the expression of TTR mRNA and TTR protein, respectively. Two F0-generation, 2 F1-generation and 15 F3-generation mice were obtained. The gene sequencing of F3 mice showed TTR Gly83Arg mutation. When examined with Congo red and polarized light, the vitreous of TTR Gly83Arg mutant mice tested positive for amyloid. The hearts, livers, brains and kidneys of the experimental group and control group were all negative by Congo red staining. Immunohistochemical staining showed that the vitreous of TTR Gly83Arg mutant mice and the livers of the control mice were positive, but the kidneys, hearts and brains of both groups were negative. Quantitative fluorescent PCR showed that the mRNA expression of mutant mice was significantly lower than that of wild-type mice (F = 0.295, P = 0.023). Western blotting showed that the expression of TTR protein was significantly lower in the model mice than in the wild-type mice (t = 3.224, P = 0.018). TTR gene mutation is indeed a molecular characteristic of HVA and manifest in the eye disease only. A C57BL/6 mouse line carrying the TTR Gly83Arg gene mutation was successfully established. This strain of mice can be used for the study of HVA.
本研究旨在通过基因打靶技术构建转甲状腺素蛋白（TTR）Gly83Arg 基因突变的小鼠模型，以研究遗传性玻璃体淀粉样变性（HVA），并确认该点突变是否是 HVA 的遗传特征。构建靶向 ES 细胞的载体（pBR322-MK-TTR）。将成功转染的 ES 细胞用于囊胚注射，从而产生 F0。F0 和 Flp 小鼠交配产生 F1（TTR，Flp）小鼠，这些小鼠缺乏 neo 基因但携带 Flp 基因。F1 小鼠与 C57BL/6N 野生型小鼠交配产生 F2（TTR）小鼠。F2 小鼠相互交配产生 F3 纯合和杂合小鼠。对 F3 小鼠进行 PCR 和测序。使用刚果红染色和偏振光检测淀粉样蛋白。免疫组织化学检测组织中 TTR 的表达。定量荧光 PCR 和 Western 印迹分别用于检测 TTR mRNA 和 TTR 蛋白的表达。获得了 2 只 F0 代、2 只 F1 代和 15 只 F3 代小鼠。F3 代小鼠的基因测序显示 TTR Gly83Arg 突变。用刚果红和偏振光检查时，TTR Gly83Arg 突变小鼠的玻璃体淀粉样蛋白检测呈阳性。实验组和对照组的心脏、肝脏、大脑和肾脏用刚果红染色均为阴性。免疫组织化学染色显示 TTR Gly83Arg 突变小鼠的玻璃体和对照组小鼠的肝脏呈阳性，但两组的肾脏、心脏和大脑均为阴性。定量荧光 PCR 显示突变小鼠的 mRNA 表达明显低于野生型小鼠（F=0.295，P=0.023）。Western 印迹显示模型小鼠的 TTR 蛋白表达明显低于野生型小鼠（t=3.224，P=0.018）。TTR 基因突变确实是 HVA 的分子特征，仅表现在眼部疾病中。成功构建了携带 TTR Gly83Arg 基因突变的 C57BL/6 小鼠品系。该品系的小鼠可用于 HVA 的研究。
