摘要
目的建立非洲爪蟾驱动蛋白样蛋白2靶蛋白(TPX2)C'末端SBP-3×Flag标记的HCT116结直肠癌细胞模型。方法 PCR扩增同源臂,构建TPX2的腺相关病毒打靶载体,包装病毒后打靶HCT116结直肠癌细胞,G418和PCR筛选获得含有新霉素抗性基因的阳性细胞株,最后通过Cre病毒感染去除抗性基因,并用PCR方法筛选获得TPX2C末端SBP和3×Flag内源性双标签的HCT116结直肠癌细胞株。结果筛选获得2个含有新霉素抗性基因的细胞株,随后经Cre病毒感染得到去除新霉素抗性基因的阳性细胞克隆,并经Western blot检测验证了SBP-3×Flag基因的敲入。结论成功建立了TPX2 C'末端SBP-3×Flag标记的HCT116结直肠癌细胞模型。
Objective To develop a targeting protein for Xenopus kinesin-like protein 2（TPX2） C＇ terminal SBP-3×Flag-tagged HCT 116 cell model.Methods Homologous arms were amplified by polymerase chain reaction（PCR）,and then the adeno-associated virus（AAV）-targeting vector of TPX2 was constructed.HCT 116 cells were targeted after the viruses were packaged.Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening.Finally,the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase,and the TPX2 C＇ terminal SBP and 3×Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.Results Two positive cell clones with neomycinresistance gene were obtained by PCR screening.The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection,and the knock-in of SBP-3×Flag gene was verified by Western blot analysis.Conclusion The TPX2 C＇ terminal SBP-3×Flag tagged HCT 116 cell model was successfully established.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2011年第6期624-628,共5页
Acta Academiae Medicinae Sinicae
基金
国家重大研究计划项目(2011CB944302
2011CB944404)
国家重点实验室专项基金(2060204)
湖北省自然科学基金(2010CDB08704)~~
