CAJ | 학술논문

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。
목적：구건저갑형주기성마비상관적Cchl1a3기인R528H고입소서모형。방법장 Cchl1a3-knock-in타파재체전전염ES세포，경과G418화Ganciclovoir사선양성ES세포극륭병용PCR화DNA측서법감정。장양성ES극륭주사도소서낭배，획득감합체소서。통과잡교획득적잡합자소서여FLP소서교배번육획득거neo잡합자소서，병용PCR화DNA측서진행감정。장거neo잡합자소서교배득도순합자후대，진행생장발육등방면적관찰。결과타파재체성공전염ES세포，PCR화DNA측서법증실9개ES세포극륭발생정학적동원중조。통과현미주사획득7지감합체소서。장감합체소서교배번육적잡합자소서화FLP소서교배획득9지거neo잡합자소서，최종득도15지거neo순합자소서。해소서재발육지성성숙계단，정신、음식급활동상태량호，단시재4개월령시축점출현탈모，피부파궤심지사망。결론성공구건Cchl1a3기인 R528H 돌변적순합자소서，위연구인류CACNA1S기인공능화천명저갑형주기성마비발생적분자궤제전정료기출。
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 G 418 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 withtargeting 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 .