摘要
目的构建酮戊二酸脱氢酶(OGDH)基因敲除SD大鼠模型。方法设计、构建TALEN重组子,用荧光素酶活性法筛选出最有效重组子,显微注射法将质粒打入受精卵后测序鉴定。对同一时间点的野生型和基因敲除型大鼠分别进行饮食习惯、活动状态、毛发颜色及质量检测,Western Blot检测两者肝组织OGDH蛋白表达。结果成功构建了靶向OGDH基因的TALEN质粒,并筛选了最有效的TALEN重组子(OGDH-T2),成功培育出杂合子18只,成功率为22.5%,未发现OGDH-KO8纯合子的出生。质量检测显示OGDH-KO8大鼠的质量明显高于野生型,且在第10周时最为显著,增幅率达50.2%(P<0.001),KO组大鼠的OGDH蛋白表达水平显著降低,下降率为50.6%(P<0.01)。结论利用TALEN技术成功构建了OGDH基因敲除型SD大鼠模型。
Objective To develope OGDH gene knockout SD rat model by transcription activator-like effector nu- cleases (TALEN) technology. Methods Three pairs of TALEN plasmid were designed and constructed, then the transfection activity was examined in 293T cells, the TALEN pair with the most effective mutation was select- ed before the plasmid pair was applied to the fertilized rat eggs using microinjection. DNA sequencing with tissue samples from two-weeks newborn SD rat was performed. The SD rats were followed by measuring the body weight, observing food taking, running activity and fur color etc. The protein expression of OGDH was detected by Western blot. Results The TALEN plasmid pair OGDH-T2 was most effective and this plasmid pair was used in this study. In total, 80 fertilized eggs were injected with the OGDH-T2 plasmid pair and 18 rats were born, with an efficiency rate of 22.5%. However, homogeneous knockout rats were not obtained after followed a period of three generations. However, heterogeneous KO mutation OGDH-KO8 was maintained. Therefore, we followed this type of heterogeneous KO rats in this study. We discovered that OGDH-KO8 influenced the body-weight.Compare(t with the control rats, the rats with the OGDH-KO8 heterogeneous mutation showe(I significantly higher body weight within the 10 weeks observation( P 〈 0. 001 ) , Otherwise, there was no difference observed in this typical heterogeneous KO rats. In consistence with the bodyweight increase, the OGDH protein expression was decreased in these rats, the rates of decreased up to 50.6% ( P 〈 0.01 ) , the expression of liver OGDH protein is 50.6% less than that of WT type. Conclusions Heterogeneous OGDH gene knockout SD rat model is success- fully established.
出处
《基础医学与临床》
CSCD
2016年第8期1068-1073,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(81272824)