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首页> 外文期刊>Molecular and Cellular Biology >Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.
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Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.

机译：大肠杆菌基因用于天冬酰胺合成酶作为穿梭载体中的选择标记，该穿梭载体能够在动物细胞中显性转染和扩增。

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摘要

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.
机译：通过将含有克隆的天冬酰胺合成酶（AS）大肠杆菌基因的2碱基碱基的基因组片段插入pBR322-猿猴病毒40重组载体pSV2中，开发了一种用于细菌-动物细胞穿梭载体的新型显性可选择性扩增系统。为了将细菌AS的翻译起始密码子置于猿猴病毒40早期启动子下游约1,000个碱基对。这种新的构建体pSV2-AS保留了用于转录和翻译起始的细菌序列，因此可以在细菌中表达AS。该构建体还可以补充哺乳动物细胞的AS-突变体，从而使AS +转染子能够在缺乏天冬酰胺的培养基中以相对较高的效率生长（每暴露10（6）个细胞每微克DNA约300个菌落）。通过在含有增加浓度的AS抑制剂β-天冬氨酰异羟肟酸酯的无天冬酰胺的培养基中进行选择，可以在这种AS +转染子中将载体扩增多达100倍。发现AS +转染子对第二种AS抑制剂Albizziin的抵抗力要比正常的AS +动物细胞系高。利用这种差异可能表明细菌AS酶对Albizziin有较强的抵抗力，从而开发出了对大鼠，中国仓鼠，小鼠和人类等多种野生型AS +细胞系的细菌AS转染子的有效选择。用pSV2-AS转染LR-73细胞（中国仓鼠AS +细胞系），效率为每10（6）个细胞每0.5微克DNA约1,000个菌落。通过将转染子在浓度递增的β-天冬氨酰异羟肟酸酯中孵育来扩增这些细胞中整合的构建体。讨论了这种新的显性，选择性和可扩增标记相对于穿梭载体中常用标记的优缺点。

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