心肌乙酰胆碱敏感钾通道基因敲除小鼠模型的建立

Establishment of acetylcholine-sensitive K~+ channels gene-knockout mouse model

关键词:  心衰  乙酰胆碱敏感性钾通道  基因打靶  胚胎干细胞  转基因小鼠  模式动物

Key words:  heart failure  acetylcholine-sensitive K~+ channels  gene target  embryo stem cells  transgenic mouse  model animal  

摘要:

目的建立Kcnj5基因敲除小鼠模型，构建病理生理或药理实验平台，以确定钾通道和东莨菪碱等药物和心衰治疗的关系。方法用细菌人工染色体（bacterial artificial chromosome, BAC）载体构建了缸面5打靶载体，电穿孔到129／S6／SvEv品系雄性小鼠的胚胎干细胞SCR012后，用300μg／mlG418、2μmol／LGanC筛选克隆8d。打靶后的干细胞显微注射到囊胚后．生育嵌合体再交配繁育杂合子及纯合子。用PCR方法结合片段克隆后基因测序鉴定基因型。免疫组化法证实基因型的表达。结果挑取抗性克隆96个中正确同源克隆数20个，阳性率为20．8％。显微注射C57BL／6J囊胚180枚．14只受体出生12只小鼠中得到4只大于50％嵌合率雄鼠。配育获得29只尉r3．4 1代杂合子。现繁育F4后代获得尉r3．4＇纯合子约百只。用单抗对敲除鼠心进行免疫组化鉴定，提示纯合子敲除鼠心为阴性。结论用同源重组法敲除了干细胞靶基因，成功建立腼r3．4‘建立鼠系。成功建立鲋r3．4钾通道分子病动物模型。该基因的完全缺如模型有利于隔离了该基因的作用，对与之相关的基因的进一步阻断、拈抗或激活等药物研究．也是一个很好的模型。

Abstract:

Objective To study the relationship between Scopolamine and K+ channels with congestive heart failure by establishing Kcnj5 gene knockout mouse model. Methods Bacterial artificial chromosome (BAC) vector was used for knockout of Kcnj5 . ES cells of 129/S6/SvEv strain S CRO 1 2 were electrotransporated and selected by 300 |!g/ml G4 1 8 and 2 |xmol/L GanC for 8 days . Then, they were micro-injected into blastula of C-57BL/6J mice. Heterozygote and homozygote were multiplied by procreation mosaic. Progeny gene type and gene expression was assayed by PCR, se-quencing and immunohistochemistry methods. Results There were 20 right homorecombinants in 96 anti-drug clones of stem cells (positive rate 20.8%). A total of 180 blastulae were manipulated while 12 of puppies were bom in some of 14 receptors. Four males were chimera which were more than 50% ratio . 29 of Kir3.4+/- F 1 heterozygote were founded. At last, hundreds of mice Kir3. 4-/-grew up, according to gene type as s ay . Conclusion Target gene of stem cell is knocked out by homologous recombination and KirSA-/-mouse strain is established successfully. It is a good animal model for blockage, antagonism and activation medicine research.