Codon optimized neomycin resistance (neo4) gene in a replicative plasmid increases the transformation efficiency of conjugative Tetrahymena thermophila by electroporation

Today, efficiency of conjugant electrotransformation (CET) of Tetrahymena thermophila with a replicative plasmid containing a paromomycin-resistant gene is still known as ~1-11% range, which is not high enough for the efficient production of germline or vegetative transgenic strains to screen cDNA or genomic DNA library. The goal of this study is to determine the efficiency of CET by using a replicative plasmid containing a paromomycin-resistant codon optimized gene (neo4) and a C3 ribosomal DNA (rDNA) replication origin. In this study, the 3.8 kb C3 rDNA replicative origin from pH4T2 were amplified and ligated to HindIII-SalI site of pNeo4 vector to create pNeo4-ori. Conjugating cells of the ciliate T. thermophila were electroporated with 15 µg of pNeo4-ori. Transformed cells were selected with paromomycin following ~12-24 hr of growth on SPP medium. We observed resistant cells in first 2-3 days until 7th day under increasing paromomycin (100, 300 and 600 µg/ml) with CdCI2 (final concentration 1 µg/ml) or without CdCI2. These results indicate that the electroporation of T. thermophila with the pNeo4-ori constructed plasmid containing C3 replicative origin and neo4 gene cassette is duable.