GENERATION OF MICE CARRYING A MUTANT APOLIPOPROTEIN-E GENE INACTIVATED BY GENE TARGETING IN EMBRYONIC STEM-CELLS
GENERATION OF MICE CARRYING A MUTANT APOLIPOPROTEIN-E GENE INACTIVATED BY GENE TARGETING IN EMBRYONIC STEM-CELLS
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DOI：
10.1073/pnas.89.10.4471
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发表时间：
1992-05-15
影响因子：
11.1
通讯作者：
MAEDA, N
中科院分区：
文献类型：
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作者：
PIEDRAHITA, JA;ZHANG, SH;MAEDA, N
我们利用基因打靶技术在小鼠胚胎干细胞中灭活了内源性载脂蛋白E（apoE）基因。使用了两种靶向质粒PJPB 63和PNMC 109，它们都含有新霉素抗性基因，该基因取代了apoE基因的一部分并破坏了其结构。用质粒pJPB 63电穿孔后靶向的ES细胞集落通过聚合酶链反应（PCR）进行鉴定，然后进行基因组Southern分析。在648个G418抗性菌落中，9个在PCR扩增后给出阳性信号，其中5个通过Southern印迹分析确认为靶向。第二个质粒pNMC 109除了新霉素抗性基因外还含有负选择性胸苷激酶基因。用该质粒电穿孔后，分析了177个对G418和更昔洛韦均具有抗性的菌落; 39个菌落含有通过Southern印迹测定的破坏的apoE基因。通过用6种靶向细胞系进行胚泡注射来产生嵌合小鼠。其中一个品系产生了强烈的嵌合体，其中三个将破坏的apoE基因传递给了它们的后代。被破坏基因的纯合子小鼠是从杂合子中产生的;它们看起来很健康，尽管它们的血浆中没有载脂蛋白E。
We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, PJPB63 and PNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.