ten Asbroek A L, Ouellette M, Borst P
Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam.
Nature. 1990 Nov 8;348(6297):174-5. doi: 10.1038/348174a0.
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 transformation allowing the short-term expression of genes reintroduced into parasites like Trypanosoma brucei, the causative agent of African trypanosomiasis. We have obtained long-term stable transformants of T. brucei that have acquired the ability to grow in medium containing the drug G418, following the targeted insertion of the bacterial gene for neomycin phosphotransferase (neo(r) gene) into the trypanosome tubulin cluster. Plasmids in which part of the T. brucei tubulin gene cluster has been replaced by the neo(r) gene were used.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 these neo(r) constructs homologous recombination seems to be the preferred route for insertion of exogenous DNA into the trypanosome genome, allowing gene targeting without counter-selection.
动质体是单细胞真核生物，包括对人类重要的寄生虫，如锥虫和利什曼原虫。对这些生物体的研究因瞬时转化技术的发展而得到了新的推动，该技术允许将重新导入寄生虫（如非洲锥虫病的病原体布氏锥虫）的基因进行短期表达。我们已经获得了布氏锥虫的长期稳定转化体，在将细菌新霉素磷酸转移酶基因（neo(r)基因）靶向插入锥虫微管蛋白基因簇后，这些转化体获得了在含有药物G418的培养基中生长的能力。使用了其中部分布氏锥虫微管蛋白基因簇被neo(r)基因取代的质粒。线性化构建体的靶向效率高于环状构建体，含有5千碱基微管蛋白基因簇的构建体的靶向效率高于含有0.9千碱基的构建体。使用这些neo(r)构建体时，同源重组似乎是将外源DNA插入锥虫基因组的首选途径，从而允许在无需反选择的情况下进行基因靶向。
