2 DOMINANT-ACTING SELECTABLE MARKERS FOR GENE-TRANSFER STUDIES IN MAMMALIAN-CELLS
2 DOMINANT-ACTING SELECTABLE MARKERS FOR GENE-TRANSFER STUDIES IN MAMMALIAN-CELLS
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DOI：
10.1073/pnas.85.21.8047
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发表时间：
1988-11-01
影响因子：
11.1
通讯作者：
MULLIGAN, RC
中科院分区：
文献类型：
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作者：
HARTMAN, SC;MULLIGAN, RC
我们报告了两个显性遗传标记的发展，用于监测哺乳动物细胞中的基因转移是基于原核基因编码的关键步骤中的必需氨基酸，色氨酸和组氨酸的合成。在适当的条件下，这些基因的表达消除了它们各自氨基酸产物的营养需求。大肠杆菌的trpB基因的表达，该基因编码β色氨酸合酶亚基（EC 4.2.1.20），允许哺乳动物细胞在含有吲哚代替色氨酸的培养基中存活和增殖。鼠伤寒沙门氏菌的hisD基因编码组氨醇脱氢酶（EC 1.1.1.23），其催化L-组氨醇两步NAD+依赖性氧化为L-组氨酸。在缺乏组氨酸和含有组氨醇的培养基中，只有表达hisD产物的哺乳动物细胞存活。该选择是双重选择，因为所提供的前体组氨醇本身通过其对组氨酰-tRNA合成酶的抑制而对动物细胞有毒;因此，脱氢酶既去除抑制剂又形成所需的终产物。或者，his选择可以在脱氢酶主要用于使组氨醇解毒的条件下进行。对于色氨酸或组氨酸的选择，替代营养素（吲哚或组氨醇）是容易获得的、廉价的、稳定的、对细胞可渗透的，并且在由单个基因控制的步骤中转化为最终产物。基于鼠逆转录病毒和乳多空病毒骨架的载体已经成功地用于这两种基因，允许在一系列细胞类型中进行选择，包括3 T3、CV-1、a和HeLa。这些占主导地位的选择方案应该为目前使用的其他方案，如gpt、neo、hygro、dhfr和tk选择方案，提供普遍有用和廉价的替代方案。
We report the development of two dominant-acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on prokaryotic genes encoding key steps in the synthesis of the essential amino acid, tryptophan and histidine. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the trpB gene of Escherichia coli, which encodes the .beta. subunit of tryptophan synthase (EC 4.2.1.20), allows mammalian cell survival and multiplication in medium containing indole in place of tryptophan. The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyzes the two-step NAD+ -dependent oxidation of L-histidinol to L-histidine. In medium lacking histidine and containing histidinol, only mammalian cells expressing hthe hisD product survive. The selection is a double one in that the provided precursor histidinol is itself toxic to animal cells through its inhibition of histidyl-tRNA synthetase; thus, the dehydrogenase both removes an inhibitor and forms a required end product. Alternatively, the his selection may be carried out under conditions in which the dehydrogenase serves mainly to detoxify histidinol. For either the trp or his selections the substitute nutrient (indole or histidinol) is readily available, inexpensive, stable, permeable to cells, and convertible to the end product in a step controlled by a single gene. Vectors based upon murine retrovirus and papovavirus backbones have been successfullly employed for both genes, allowing selection in a range of cell types, including 3T3, CV-1, aand HeLa. These dominant selective schemes should provide generally usefula and inexpensive alternatives to others currently in use, such as the gpt, neo, hygro, dhfr, and tk selection.