Martínez-Calvillo S, López I, Hernández R
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D.F.
Gene. 1997 Oct 15;199(1-2):71-6. doi: 10.1016/s0378-1119(97)00348-x.
To improve the selection phenotype of the expression plasmid pTEX, a Trypanosoma cruzi rDNA (DNA coding for rRNA) gene spacer fragment (806 bp) containing a mapped transcription start point (tsp) was cloned in the vectors pTEX and pTEX-cat, generating the plasmids pRIBOTEX and pRIBOTEX-cat. T. cruzi cultures transiently transfected with pRIBOTEX-cat expressed a chloramphenicol (Cm) acetyltransferase (CAT) activity 16,000-fold greater than the activity observed with the parental vector pTEX-cat. Moreover, T. cruzi cells transformed with pRIBOTEX and pRIBOTEX-cat exhibited logarithmic growth in the presence of Geneticin (G418) 2 weeks earlier than that observed with controls transformed with pTEX. The plasmid copy number in stably transformed trypanosomes was about 50-times higher in cultures transformed with pTEX-cat than in cells transformed with pRIBOTEX or pRIBOTEX-cat. However, the neo RNA steady-state level and the CAT activity observed among the stably transfected cultures showed only modest differences. Finally, it was found that the pRIBOTEX vector was not episomally maintained as pTEX, but integrated into a chromosome indistinguishable from the one encoding rRNA. These features make pRIBOTEX a useful tool for transfection and rapid expression of genes in T. cruzi.
为了改善表达质粒pTEX的选择表型，将含有一个已定位转录起始点（tsp）的克氏锥虫rDNA（编码rRNA的DNA）基因间隔区片段（806 bp）克隆到载体pTEX和pTEX-cat中，构建了质粒pRIBOTEX和pRIBOTEX-cat。用pRIBOTEX-cat瞬时转染的克氏锥虫培养物表达的氯霉素（Cm）乙酰转移酶（CAT）活性比亲本载体pTEX-cat所观察到的活性高16000倍。此外，用pRIBOTEX和pRIBOTEX-cat转化的克氏锥虫细胞在含有遗传霉素（G418）的情况下比用pTEX转化的对照细胞提前2周呈现对数生长。在用pTEX-cat转化的培养物中，稳定转化的锥虫中的质粒拷贝数比用pRIBOTEX或pRIBOTEX-cat转化的细胞中的质粒拷贝数高约50倍。然而，在稳定转染的培养物中观察到的新霉素RNA稳态水平和CAT活性仅显示出适度差异。最后，发现pRIBOTEX载体不像pTEX那样以附加体形式维持，而是整合到一条与编码rRNA的染色体无法区分的染色体中。这些特性使pRIBOTEX成为在克氏锥虫中转染和快速表达基因的有用工具。
