Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.
Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.
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删除 PGK-新霉素抗性后，猪胎儿成纤维细胞中人源化 Fat-1 的表达降低。
DOI：
10.4238/2015.september.28.11
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发表时间：
2015-09
期刊：
影响因子：
--
通讯作者：
Li X L
中科院分区：
文献类型：
--
作者：
Liang H;Han X J;Yun T;Zhao Y H;Zhang M L;Zhao L H;Li R F;Li X L
新霉素抗性基因（neo（r））作为一种选择标记被广泛应用于真核表达载体中，但其表达常常影响靶基因的表达。Cre重组酶识别LoxP位点，导致两个LoxP位点之间的DNA和RNA的位点特异性重组和缺失。本研究利用DNA Works技术获得人源化的Fat-1基因（hFat-1），并构建pC-PGK-neo（r）-hFat-1表达载体。用脂质体将pC-PGK-neo（r）-hfat-1质粒转染猪胎儿成纤维细胞，用400 μg/mL G418培养7 d，获得3株转基因细胞系。接着，用含有嘌呤霉素抗性基因的Cre重组酶表达质粒转染这些细胞系，以删除整合到基因组中的neo（r）。在嘌呤霉素选择后获得hFat-1-neo（r）阴性细胞。实时定量聚合酶链反应数据表明，新霉素耐药细胞hFat-1的表达高于新霉素敏感细胞。高效气相色谱数据表明，n-6/n-3的比例显着低于在野生型细胞中的转染细胞。Cre处理的hFat-1转染细胞中的n-6/n-3比值高于未处理的细胞，表明PGK-neo（r）的缺失降低了hFat-1的表达。
The neomycin-resistance (neo(r)) gene is widely used as a selectable marker in eukaryotic expression vectors; however, its expression often affects that of target genes. Cre recombinase recognizes LoxP sites, leading to site-specific recombination and deletion of DNA and RNA between two LoxP sites. In the present study, a humanized Fat-1 gene (hFat-1) was generated by DNA Works and used to construct a pC-PGK-neo(r)-hfat-1 expression vector, in which PGK-neo(r) was flanked by two LoxP sites. The pC-PGK-neo(r)-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 μg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neo(r), which was integrated into the genome. hFat-1-neo(r) negative cells were obtained following puromycin selection. Real-time quantitative polymerase chain reaction data indicated that neomycin-resistant cells had higher hFat-1 expression than neomycin-sensitive cells. High performance gas chromatography data suggested that the n-6/n-3 ratio was significantly lower in transfected cells than in wild-type cells. The n-6/n-3 ratio in Cre-treated hFat-1-transfected cells was higher than that in untreated cells, suggesting that deletion of PGK-neo(r) decreased hFat-1 expression.
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影响因子：
4.8
作者：
M. Holzenberger;P. Leneuve;G. Hamard;B. Ducos;L. Périn;M. Binoux;Y. Bouc
通讯作者：
M. Holzenberger;P. Leneuve;G. Hamard;B. Ducos;L. Périn;M. Binoux;Y. Bouc
影响因子：
14.8
作者：
Davis, Richard P.;Costa, Magdaline;Stanley, Edouard G.
通讯作者：
Stanley, Edouard G.
影响因子：
9.1
作者：
Pan DengKe;Zhang Li;Chen HongXing
通讯作者：
Chen HongXing
影响因子：
10.5
作者：
FIERING, S;EPNER, E;GROUDINE, M
通讯作者：
GROUDINE, M
影响因子：
46.9
作者：
Lai, LX;Kang, JX;Dai, YF
通讯作者：
Dai, YF