Yancey R J, Kotarski S F, Thurn K K, Lepley R A, Mott J E
Upjohn Company, Kalamazoo, MI.
J Ind Microbiol. 1993 Jul;11(4):259-71. doi: 10.1007/BF01569599.
Strain BST-1 is a derivative of Escherichia coli K-12 that carries a plasmid designated pURA-4 and is the expression system used by The Upjohn Company in the production of recombinant bovine somatotropin (rbSt). This plasmid also encodes an ampicillin resistance gene. The plasmidless carrier strain, BST-1C, contains a gene for tetracycline resistance which is provided by the chromosomal insertion of the transposon Tn10. Therefore, BST-1 is resistant to ampicillin and tetracycline, while BST-1C is resistant only to tetracycline. The Food and Drug Administration requested that we conduct an environmental assessment study to monitor the 'persistence of the recombinant live K-12 E. coli organism compared to the host E. coli organism'. In addition, we were requested to monitor 'the potential transfer of genetic material from (our) recombinant organism to the indigenous microflora' of the mouse gastrointestinal (GI) tract. The differences in persistence were determined by monitoring shedding of BST-1 and BST-1C in the feces of conventionally reared, outbred mice inoculated with either of the two strains. Even with antibiotic selective pressure applied (tetracycline in the water), BST-1 did not persist as well as the non-plasmid carrying parental stain, BST-1C. In the gene transfer experiments, transfer of pURA-4 was monitored by the appearance of the ampicillin resistance marker and/or by hybridization assays for the rbSt gene in indigenous, mouse-colonizing E. coli strains which had been made streptomycin resistant. At the limit of detection, no transfer of pURA-4 was detected either in vitro or in vivo. These data support an interpretation that BST-1 does not present an environmental hazard as measured by colonization/persistence in the gut of conventionally reared mammals.
菌株BST - 1是大肠杆菌K - 12的衍生物，携带一个名为pURA - 4的质粒，是Upjohn公司用于生产重组牛生长激素（rbSt）的表达系统。该质粒还编码氨苄青霉素抗性基因。无质粒载体菌株BST - 1C含有一个四环素抗性基因，该基因由转座子Tn10的染色体插入提供。因此，BST - 1对氨苄青霉素和四环素具有抗性，而BST - 1C仅对四环素具有抗性。美国食品药品监督管理局要求我们进行一项环境评估研究，以监测“重组活K - 12大肠杆菌与宿主大肠杆菌相比的持久性”。此外，要求我们监测“遗传物质从（我们的）重组生物体向小鼠胃肠道（GI）道的本土微生物群的潜在转移”。通过监测接种了这两种菌株之一的常规饲养的远交系小鼠粪便中BST - 1和BST - 1C的排出情况来确定持久性差异。即使施加抗生素选择压力（水中添加四环素），BST - 1的持久性也不如无质粒的亲本菌株BST - 1C。在基因转移实验中，通过氨苄青霉素抗性标记的出现和/或对已产生链霉素抗性的本土小鼠定殖大肠杆菌菌株中的rbSt基因进行杂交分析来监测pURA - 4的转移。在检测限内，无论是在体外还是体内均未检测到pURA - 4的转移。这些数据支持这样一种解释，即按照在常规饲养哺乳动物肠道中的定殖/持久性来衡量，BST - 1不会造成环境危害。
