不同条件下人乳白蛋白YAC的高效定点打靶修饰

Site-specific modification of human α-lactalbumin yeast artificial chromosome by targeting under different conditions

目的?对人乳白蛋白酵母人工染色体(HLA-YAC)进行定点改造,以便进一步利用该载体作与基因表达调控相关的 研究。方法:借助kar1突变使YPH925菌株与异交配型菌株交配时丧失核融合的能力,把HLA-YAC由酵母AB1380转移到 his3缺陷的YPH925菌株。分别以HIS3(对YPH925菌株)和G418R(对YPH925和AB1380两种菌株)为选择标记,用含 HLA-YAC的酵母基因组为模板,PCR克隆人乳白蛋白(HLA)基因的5′和3′端非翻译区各684bp和940bp为同源臂,构建 打靶载体,线性化的打靶载体经电转化或醋酸锂转化转入酵母内,以菌落PCR为鉴定方法,辅以测序验证。结果:HLA-YA 在目的位置完成了定点打靶修饰,G418R的筛选效率高于HIS3,醋酸锂转化方法的打靶效率稍高于电转化。结论:对YA 的成功修饰为在动物体内的表达及调控研究打下了基础。

Objective:To modify human α-lactalbumin yeast artificial chromosome(HLA-YAC)and to use it as vectors for analysis of gene expression and regulatory elements of α-lactalbumin in vivo in the future.Methods:In virtue of kar1mu-tants,YPH925lost the ability of nuclear fusion when it mated with opposite mating type strain.HLA-YAC was transferred from donor yeast strain AB1380to recipient his3-deficient strain YPH925.Two homologous arms were cloned from YAC by PCR:940bp and680bp from5′and3′untranslated region of α-lactalbumin gene,respectively.Yeast dominant selectable marker(G418R)was placed in the pRS403containing HIS3as the targeting vector.Linearized targeting vector was intro-duced into yeasts by transformation of lithium acetate or electroporation.The clones were analyzed using PCR and sequence detection.Results:HLA-YAC was modified by gene targeting.The efficiency of G418R selection was significantly higher than that of HIS3,and positive efficiency by lithium acetate transformation was a little higher than that by electroporation.Conclusion:The production of the modified HLA-YAC provides a foundation for investigation of gene expression and analysis of the regulatory elements in vivo. [