Tetrahymena thermophila, Nanney & McCoy, 1976

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 : 8-9

Gene overexpression in T. thermophila

Overexpression is a commonly used technique in biological research and can be applied to produce economically valuable products (David-Pfeuty and Nouvian-Dooghe 1990, Prelich 2012, Wang et al. 2019).

In T. thermophila , ribosomal DNA (rDNA) exists as a single copy in the MIC chromosome and is flanked by a chromosome breakage sequence (Yao and Gall 1977). During new MAC development in conjugation, the rDNA region is excised from the chromosome and rearranged into a 21 kb inverted repeat, termed an rDNA palindrome, then amplified to a copy number of ~10 000 ( Brunk 1986). The use of rDNA or plasmids containing rDNA replication origins as vectors to carry the target gene allows the target gene to be introduced into the MAC and maintain its high copy number (Table 1). The constructed DNA is integrated into the rDNA mini-chromosome and transcribed normally. Expression of the transformed target gene can be regulated further by modifying its promoter, such as by replacing the original promoter with the histone H4 promoter (Yao and Yao 1991, Kahn et al. 1993, Gaertig and Gorovsky 1995).

Another way to promote gene overexpression in T.thermophila is to use homologous recombination to replace a non-essential gene with the target gene (Table 1; Qiao et al. 2017, Wang et al. 2019). The most frequently replaced genes are the expressioninducible genes MMT1 and MMT3 ( Fig. 5A ). This is very similar to the MAC gene knockout strategy described above, except that both MMT1 and MMT3 are replaced, with the target gene and neo Tet, respectively. It is noteworthy that the drug-resistance gene used here is only the coding region rather than the whole cassette of neo4, because expression of neo Tet is regulated by the MMT3 promoter ( Figs 2A , 5A).

Replacing non-essential genes (MMT1 and MMT3) with a target gene, coupled with the regulation of overexpression levels and timing by the addition and removal of Cd 2+, offers greater control in comparison to increasing the gene copy number through rDNA-dependent replication ( Boldrin et al. 2006).

Figures

Figure 2. Gene knockout based on homologous recombination in Tetrahymena thermophila. A* schematic diagram of neo4 selectable casseưe (a) and flow path of homologous recombination (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR.Red arrows pointing at the plasmid indicate the restriction enzyme digestion sites. B* cell screening of macronuclear gene knockout. A cross covering the cell means that the cell is dead. Ŋe black bars represent the endogenous chromosomes* and pink rectangles indicate the exogenous DNA that has been integrated into the chromosomes. C* procedures for micronuclear gene knockout in Tetrahymena. Ŋe correspondence between genotypes and cell icons is listed in the key at the boưom less. Abbreviations:UºR* untranslated regions; BTU2* a gene encoding β-tubulin; GOI* gene of interest; MAC* macronucleus; MIC* micronucleus; 6-MP* 6-methylpurine; MT1* a cadmiuminducible metallothionein gene; neo Tet* a synthetic neo gene (neomycin-resistance gene) optimized for codon usage of Tetrahymena; PM* paromomycin.

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.