Oxytricha trifallax, Greslin et al., 1989

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 : 10

Gene overexpression in O. trifallax

Benefitting from the gene-sized nano-chromosomes in the macronuclear genome, gene overexpression in O. trifallax can be achieved by introduction of synthetic artificial chromosomes (Table 1; Swart et al. 2013). Clay et al. (2019a) successfully transferred an artificial chromosome, comprising complete flanking sequences, a target gene, and 20 bp of telomere sequences at each end, into the MAC of vegetative cells by microinjection to obtain functional proteins. The injected chromosomes were stably maintained in cells (over 40 generations) and were largely free of end erosion, even after encystment/excystment ( Clay et al. 2019a). It is noteworthy that the introduction of synthetic DNA homologous to an endogenous nano-chromosome into O. trifallax during gene rearrangement resulted in decreased rather than increased expression of the corresponding genes, suggesting that the import of artificial chromosomes for overexpression should be at the vegetative stage ( Clay et al. 2019b).

Artificial chromosome transformation has also been reported in Euplotes and Stylonychia (Table 1), where most chromosomes have the same gene size as Oxytricha (Swanton et al. 1980) . In Euplotes crassus Dujardin , 1841, artificial chromosomes were transferred into the newly developed MAC (72–75 h after forming pairs) by microinjection ( Erbeznik et al. 1999) and into vegetative cells with the aid of non-liposomal lipid Effectene (Qiagen, Hilden, Germany) ( Bender et al. 1999); in both cases, the introduced DNA could be stably maintained and expressed ( Bender et al. 1999, Erbeznik et al. 1999). In Stylonychia lemnae , injected artificial chromosomes could be stably maintained for ≥80 generations, with expression similar to that of the endogenous genes (Skovorodkin et al. 1999, 2001).

In Oxytricha , Euplotes , and Stylonychia , the artificial chromosome can replicate as efficiently as endogenous ones ( Bender et al. 1999, Skovorodkin et al. 2001, Clay et al. 2019a); however, unlike Paramecium , telomere sequences are essential to them, and the construct without telomeres is degraded soon after being injected into Stylonychia lemnae (Skovorodkin et al. 2001) .

Compared with gene knockout and knockdown, gene overexpression is highly dependent on the maintenance of introduced DNA. For species with long MAC chromosomes, the attempt can start by introducing the target gene with flanking sequences. However, for species with nano-chromosomes in the MAC, introducing the entire chromosome with telomeres is more feasible.