Bag3 P215L基因突变小鼠的繁殖与基因型鉴定

BAG3是一种多功能蛋白,在多种疾病的发生发展中起决定性作用.临床研究表明,BAG3蛋白的第209位残基脯氨酸到亮氨酸p. Pro209Leu (c. 626C T)的杂合子突变能够引起一种罕见的肌原纤维肌病.为进一步研究BAG3 P209L突变引起的相关疾病,建立了Bag3 P215L突变的小鼠模型(小鼠的Bag3蛋白序列中对应突变的脯氨酸位于第215位残基上).将Bag3 P215L杂合突变的雄鼠和雌鼠进行繁殖交配,将得到纯合子、杂合子及野生型3种基因型小鼠.出生后3～4周龄的小鼠剪尾,采用了NaOH法、KCl法和改良SDS法共3种方法提取基因组DNA,然后进行PCR扩增、琼脂糖凝胶电泳和DNA测序.用改良SDS法能更为稳定有效地提取DNA,从而成功进行Bag3 P215L小鼠的繁育和基因型鉴定.为进一步研究BAG3 P209L突变引起的相关疾病提供了理想的动物模型.

Reproduction and genotype identification of Bag3 P215L knock-in mice

BAG3 is a multifunctional protein which plays a decisive role in the development of many diseases. The single missense mutation of proline to leucine at residue 209(p.Pro209 Leu) of BAG3(c.626 C > T) causes a severe type of myofibrillar myopathy(MFM). In order to study the related diseases caused by BAG3 P209 L mutation, we generated Bag3 P215 L knock-in mice(The proline in the Bag3 protein sequence of the mouse is located on the 215 th residue). By mating between the heterozygous mutant mice, three genotypes of new born mice, homozygote, heterozygote and wild type, were obtained. Three methods, including NaOH, KCl and SDS, were used to extract genomic DNA from the mouse tail; then, the 506 bp target gene was amplified with PCR and identified by DNA sequencing. The protocol which yielded a highly purified DNA preparation was more efficient. After reproduction, the genotypes of the new born mice were successfully identified with this method. It provides an ideal animal model for further study of the related diseases caused by BAG3 P209 L mutation.