Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.
Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.
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通过基因靶向修饰 HepG2 细胞中的载脂蛋白 B 基因。
DOI：
10.1172/jci115845
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Young,SG
中科院分区：
文献类型：
--
作者：
FareseJr,RV;Flynn,LM;Young,SG
HepG2 细胞系已被广泛用于研究载脂蛋白 (apo) B 的合成和分泌。在本研究中，我们测试了基因打靶技术是否可用于通过使用转染的基因打靶载体进行同源重组来灭活 HepG2 细胞中的 apo B 等位基因之一。我们的载体含有 apo B 基因的外显子 1-7，其中外显子 2 被无启动子的新霉素抗性 (neo(r)) 基因中断。该载体与同源基因的重组将使apo B等位基因失活，并使apo B启动子能够激活neo(r)基因的转录。为了检测罕见的同源重组克隆，我们开发了一种新型固相 RIA，它使用 apo B 特异性单克隆抗体 MB19 来分析 G418 抗性 (G418r) 克隆分泌的 apo B。抗体 MB19 通过分别以高亲和力和低亲和力与 apo B 同种异型 MB19(1) 和 MB19(2) 结合，检测 apo B 中的两个等位基因遗传多态性。 HepG2 细胞通常分泌 apo B MB19 同种异型。使用 MB19 免疫测定，我们鉴定了一个 G418r HepG2 克隆，该克隆已失去分泌 MB19(1) 同种异型的能力。通过聚合酶链式反应扩增目标 apo B 等位基因特有的 865 bp 片段以及基因组 DNA 的 Southern 印迹，证实该克隆的 apo B 等位基因失活。这项研究表明，基因靶向技术可用于修饰 HepG2 细胞中的 apo B 基因，并证明了新型固相 RIA 系统在检测该细胞系中 apo B 基因靶向事件方面的有用性。
The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line.Images