低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型的构建

目的构建低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型。方法将Cchl1a3-knock-in打靶载体电转染ES细胞,经过G418和Ganciclovoir筛选阳性ES细胞克隆并用PCR和DNA测序法鉴定。将阳性ES克隆注射到小鼠囊胚,获得嵌合体小鼠。通过杂交获得的杂合子小鼠与FLP小鼠交配繁育获得去neo杂合子小鼠,并用PCR和DNA测序进行鉴定。将去neo杂合子小鼠交配得到纯合子后代,进行生长发育等方面的观察。结果打靶载体成功转染ES细胞,PCR和DNA测序法证实9个ES细胞克隆发生正确的同源重组。通过显微注射获得7只嵌合体小鼠。将嵌合体小鼠交配繁育的杂合子小鼠和FLP小鼠交配获得9只去neo杂合子小鼠,最终得到15只去neo纯合子小鼠。该小鼠在发育至性成熟阶段,精神、饮食及活动状态良好,但是在4个月龄时逐渐出现脱毛,皮肤破溃甚至死亡。结论成功构建Cchl1a3基因R528H突变的纯合子小鼠,为研究人类CACNA1S基因功能和阐明低钾型周期性麻痹发生的分子机制奠定了基础。

Construction of Cchl1a3 Gene R528H Knock-in Mouse Model Related to Hypokalemic Periodic Paralysis

Objective To construct Cchl1a3 gene R528H knock-in mouse model related to hypokalemic periodic paralysis.Methods ES cells were transfected with Cchl1a3-Konckin targeting vector linearized by Not I digestion, selected in the medium containing both G418 and ganciclovoir.Resistant clones were screened by PCR and further confirmed by DNA sequencing for correct homologous recombinants.Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. Heterozygous mice were obtained by mating. Through heterozygous mice with FLP mice mating, removal of neo gene heterozygous mice were established and identified with the PCR and DNA sequencing. After mating, homozygous offspring were constructed and observed.Results ES cells were successfully transfected with targeting vector. It was confirmed that 9 resistant clones happened right homologous recombination by PCR and DNA sequencing. 7 chimera mice were obtained by microinjection. After breeding the chimeric mice, heterozygous mice were mated FLP mice to obtain 9 heterozygous mice removal of neo gene, the finally obtained 15 homozygous mice with Flp-deleted neo gene. In the developmental stage of sexual maturity, the spirit of the mice, restaurants and activities in good condition, but the gradual emergence of hair removal at 4 months of age, skin ulceration and even death. Conclusions We successfully constructed Cchl1a3 gene R528H mutation homozygous mice. And it laid a foundation for the study of human CACNA1S gene function and to clarify the molecular mechanism of hypokalemic periodic paralysis.