TARGETED INSERTION OF THE NEOMYCIN PHOSPHOTRANSFERASE GENE INTO THE TUBULIN GENE-CLUSTER OF TRYPANOSOMA-BRUCEI
TARGETED INSERTION OF THE NEOMYCIN PHOSPHOTRANSFERASE GENE INTO THE TUBULIN GENE-CLUSTER OF TRYPANOSOMA-BRUCEI
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DOI：
10.1038/348174a0
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发表时间：
1990-11-08
期刊：
影响因子：
64.8
通讯作者：
BORST, P
中科院分区：
文献类型：
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作者：
TENASBROEK, ALMA;OUELLETTE, M;BORST, P
KINETOPLASTIDS是单细胞真核生物，包括人类重要的寄生虫，如锥虫和利什曼原虫。对这些生物的研究最近得到了短暂转化1 - 5的发展，这种转化1 - 5允许将基因的短期表达重新引入寄生虫，如非洲锥虫病的病原体布鲁氏锥虫3 - 5。我们获得了长期稳定的变压器。在将细菌基因新霉素磷酸转移酶（neorgene）靶向插入锥虫微管蛋白簇后，获得了在含有药物G418的培养基中生长的能力。质粒在t的哪个部分。布鲁氏微管蛋白基因簇已被新基因所取代。线性化的定位效率高于圆形结构，5千碱基的微管蛋白基因簇的定位效率高于0.9千碱基的定位效率。有了这些结构，同源重组似乎是将外源DNA插入锥虫基因组的首选途径，允许基因靶向而无需反选择。
KINETOPLASTIDS are unicellular eukaryotes that include important parasites of man, such as trypanosomes and leishmanias. The study of these organisms received a recent boost from the development of transient transformation1–5allowing the short-term expression of genes reintroduced into parasites likeTrypanosoma brucei3–5, the causative agent of African trypanosomiasis. We have obtained long-term stable transformants ofT. bruceithat have acquired the ability to grow in medium containing the drug G418, following the targeted insertion of the bacterial gene for neomycin phosphotransferase (neorgene) into the trypanosome tubulin cluster. Plasmids in which part of theT. bruceitubulin gene cluster has been replaced by theneorgene were used. Targeting efficiency was higher with a linearized than with a circular construct, and with 5 kilobases of tubulin gene cluster than with 0.9 kilobases. With theseneorconstructs homologous recombination seems to be the preferred route for insertion of exogenous DNA into the trypanosome genome, allowing gene targeting without counter-selection.