Ladygin V G
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Oblast, 142290 Russia.
Mikrobiologiia. 2003 Sep-Oct;72(5):658-65.
The cell wall-lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase gene hpt as the selective marker and the Tn5 transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10(6) mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10(3) HygR transformants per 10(6) recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation of Ch. reinhardtii with heterologous genes are discussed.
单细胞绿藻莱茵衣藻(Chlamydomonas reinhardtii)缺乏细胞壁的突变体CW-15通过电穿孔法用质粒pCTVHyg进行转化,该质粒构建时以潮霉素磷酸转移酶基因hpt作为选择标记,并带有受病毒SV40早期基因启动子控制的大肠杆菌Tn5转座子。在最佳条件下(10⁶个处于指数生长期中期的细胞/毫升;电场强度1 kV/厘米;脉冲长度2毫秒),转化后每10⁶个受体细胞可产生10³个潮霉素抗性(HygR)转化体。整合到莱茵衣藻核基因组中的外源DNA持续遗传了超过350个细胞世代。讨论了该系统用于用异源基因转化莱茵衣藻的优势。