A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon Tn5 under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 106 cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 103 hygromycin-resistant (HygR) clones per 106 HygR recipient cells. Exogenous DNA integrated in the nuclear genome of C. reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the HygR trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C. reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using theCW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.
Russian Journal of Plant Physiology – Springer Journals
Published: Oct 13, 2004
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