Co-transformation of Lec 1 CHO cells with N-acetylglucosaminyltransferase 1 activity and a selectable marker.
Co-transformation of Lec 1 CHO cells with N-acetylglucosaminyltransferase 1 activity and a selectable marker.
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Lec 1 CHO 细胞与 N-乙酰氨基葡萄糖转移酶 1 活性和选择标记的共转化。
DOI：
10.1002/jcb.240420302
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发表时间：
1990
影响因子：
4
通讯作者：
Fregien,N
中科院分区：
文献类型：
--
作者：
Ripka,J;Pierce,M;Fregien,N
在动物细胞中，α(1,3)‐甘露糖苷‐β(1,2)‐N‐乙酰氨基葡萄糖转移酶I （GlcNAc‐TI， EC.2.4.1.101）催化N‐乙酰氨基葡萄糖加入ASN‐连接的Man GlcNAc低聚糖。中国仓鼠卵巢（CHO）突变细胞系Lecl缺乏这种酶的活性，因此积累了甘露糖终止细胞表面ASN连接的低聚糖。因此，Lecl细胞对甘露糖结合凝集素cona （cona）的细胞毒性作用敏感。通过磷酸钙/DNA介导的转化，将来自A431细胞的人DNA和含有细菌新基因的真核表达质粒共同转化Lecl细胞。选择共变以抵抗Con A和G - 418。从初级共转化体中提取的DNA被纯化并用于转化Lecl细胞，产生二级共转化体。一级和二级共转化子均表现出体外GlcNAc - TI特异性酶活性，DNA凝胶印迹分析表明二级共转化子包含人类和新序列。
In animal cells, the enzyme α( 1,3)‐mannoside‐β( 1,2)‐N‐acetylglucosaminyltransferase I (GlcNAc‐TI, EC.2.4.1.101) catalyzes the addition of N‐acetylglucosamine to the ASN‐linked Man GlcNAc oligosaccharide. The Chinese hamster ovary (CHO) mutant cell line Lecl is deficient in this enzyme activity and, therefore, accumulates mannose‐terminating cell surface ASN‐linked oligosaccharides. Consequently, Lecl cells are sensitive to the cytotoxic effects of the mannose‐binding lectin Concanavalin A (Con A). Lecl cells were co‐transformed with human DNA from A431 cells and eukaryotic expression plasmids containing the bacterialneogene by calcium phosphate/DNA‐mediated transformation. Co‐transformants were selected for resistance to Con A and G‐418. DNA from a primary co‐transformant was purified and used to transform Lecl cells, resulting in secondary co‐transformants. Both primary and secondary co‐transformants exhibited in vitro GlcNAc‐TI‐specific enzyme activity, DNA gel blot analysis indicated that secondary co‐transformants contained both human andneosequences.