Paramecium tetraurelia, Sonneborn

Fu, Jinyu, Gao, Yunyi, Gao, Feng, Sheng, Yalan, Al-Farraj, Saleh A., Chen, Zigui & Wang, Chundi, 2024, An overview of genetic manipulation tools in the studies of ciliates * with emphasis on gene knockout * knockdown * and overexpression, Zoological Journal of the Linnean Society 202 (3), pp. 1-13 : 9

Gene overexpression in P. tetraurelia

Gene overexpression can be achieved by introducing more copies of the target gene into the MAC in P. tetraurelia (Table 1). A small portion of exogenous DNA introduced into the MAC of P. tetraurelia can be integrated into the chromosomes, similar to Tetrahymena , while most is added with Paramecium telomerelike sequences at both termini and replicates autonomously ( Gilley et al. 1988, Bourgain and Katinka 1991). Not only the linear DNA but also the circular DNA can be maintained by cleavage at non-random sites to convert it from supercoiled to linear ( Godiska et al. 1987, Gilley et al. 1988). To guarantee the efficiency of retention of exogenous DNA, linear DNA is used in routine experiments.

The flanking sequences and target gene are amplified and inserted into the vector ( Fig. 5B ). To meet the requirements of some experiments, a tag sequence can be added immediately after the start codon or before the stop codon of the target gene. The plasmid is linearized by restriction enzyme digestion at the backbone region, then introduced into the MAC of vegetative cells by microinjection. Positive injected cells can be checked using PCR or dot-blot by specifically detecting the DNA that is not present in the genome of Paramecium . Owing to the high concentration of DNA microinjected, the target gene in the transformed cells is prominently overexpressed compared with the wild-type cells. Moreover, the injected linear constructs can be maintained for a long time in vegetative cells without decreasing rapidly during cell division ( Gilley et al. 1988). However, DNA is lost with the degradation of the old MAC during conjugation.

Figures

Figure 5. Gene overexpression strategies in ciliates.A* construct of gene overexpression and the homologous recombination in Tetrahymena (a) and positive transformed cell screening (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR.Red arrows pointing to the plasmid indicate the restriction enzyme digestion sites. B* gene overexpression by injection of more copies in Paramecium. ºelomeres are indicated with red squares at the end of chromosomes. C* gene overexpression by introduction of artificial chromosomes in Oxytricha. Abbreviations:GOI* gene of interest; MAC* macronucleus; MIC* micronucleus; MT1 and MT3* cadmiuminducible metallothionein genes; neo Tet* a synthetic neo gene (neomycin-resistance gene) optimized for codon usage of Tetrahymena; PM* paromomycin.