6-磷酸果糖激酶-2／果糖双磷酸酶-2同工酶3基因敲除小鼠模型的构建

Establishment of 6 - phosphofructo - 2 - kinase/ fructose - 2,6 - biphosphatase 3 gene knockout mice

目的建立6-磷酸果糖激酶-2／果糖双磷酸酶-2同工酶3（PFKFB3）基因敲除小鼠模型。方法利用类转录激活样效应因子核酸酶（TALEN）技术，通过构建针对PFKFB3基因的TALEN质粒，并将构建好的TALEN质粒在体外反转录为mRNA。利用原核显微注射分别将0．1μl转录后的浓度为50ng／山的mRNA注射到C57BL／6品系小鼠受精卵中，将受精卵回送到ICR代孕母鼠输卵管中，得到F0代基因敲除杂合子小鼠。将FU代饲养至10周龄后合笼，从而构建基因敲除纯合子小鼠。所有子代鼠出生1周后剪尾，提取RNA后反转录，PCR产物作为模板进行基因测序鉴定基因型。结果通过TALEN技术成功构建了f1D代基因敲除杂合子小鼠3只，基因测序结果表明分别于靶基因位置缺失了10、4、1对碱基对；因PFKFB3基因敲除纯合子具有胚胎致死性，6只F1代基因敲除小鼠的基因测序结果显示，针对靶基因，编号4、5的小鼠缺失了1对碱基对，6号小鼠缺失4对碱基对，7、8、9号小鼠缺失10对碱基对。结论成功构建了PFKFB3基因敲除杂合子（＋／-）小鼠．。

Objective To generate 6 - phosphofructo -2 - kinase/fructose -2,6 - biphosphatase 3 （PFKFB3） knockout alice model. Methods The gene knockout mice were constructed by means of transcription activator - like effector nuclease （TALEN） technique. TALEN vectors were constructed targe- ting sequence of PFKFB3 and the TALEN mRNA was generated by in vitro transcription. Then 0. 1μ1 mRNA which has been diluted to 50 ng/μl was injected into fertilized C57BL/6 eggs. They were then implanted in ICR female mice for production of F0 generation knockout mouse. To generate homozygote gene knockout mice, 10 -weeks -old FO generation female mice were mated with male. The RNA was extracted from tail tips of mouse pups, then subjected to reverse transcription and the polymerase chain reaction （PCR） products were used as templates for sequencing analysis. Results By means of TALEN technique, three heterozygote PFKFB3 knockout mice （ ＋ / - ） of FO generation were obtained and sequence alignment results showed that the three mice were deleted 10 bp （No. 1 ）, 4 bp （No. 2） and 1 bp （No. 3） in target site, respectively. Due to the embryonic lethality of homozygote （ -/- ）, six heterozygote PFKFB3 knock- out mice （ ＋/- ） of F1 lines were obtained and showed that 1 bp （No. 4 and5）, 4 bp （No. 6） and 10 bp （No. 7, 8 and 9） were deleted respectively in target site. Conclusion The heterozygote PFKFB3 gene knockout mice （ ＋/- ） model has been successfully established.