High frequency vector-mediated transformation and gene replacement in Tetrahymena

Area of Science:

• Molecular Biology
• Genetics
• Protozoology

Background:

• Established methods for Tetrahymena thermophila transformation include microinjection and DNA-mediated transformation.
• Homologous recombination at the H4-I locus has been previously demonstrated using neomycin resistance genes.
• Development of efficient gene manipulation tools is crucial for Tetrahymena research.

Purpose of the Study:

• To describe the application of conjugant electrotransformation (CET) for gene replacement in Tetrahymena.
• To develop new independently replicating vectors and a selectable marker cassette for gene knockout experiments.
• To optimize transformation efficiency using replication origins and assess promoter/terminator regions.

Main Methods:

• Utilized CET to introduce DNA into conjugating Tetrahymena cells.
• Employed a neomycin resistance gene flanked by H4-I gene regulatory sequences for transformation.
• Investigated the effect of releasing the insert, replication origins (single vs. tandem repeat), and gene cassette constructs (H4-I/neo/BTU2) on transformation efficiency.

Main Results:

• CET successfully achieved H4-I gene replacement and integrative recombination.
• Releasing the insert increased gene replacement frequency six-fold.
• A tandem repeat of the rDNA replication origin significantly enhanced CET efficiency, enabling promoter mapping and the creation of a functional H4-I/neo/BTU2 selectable marker cassette.

Conclusions:

• CET is an effective method for gene replacement and developing tools for gene knockout in Tetrahymena.
• The H4-I/neo/BTU2 cassette serves as a versatile selectable marker.
• The developed vectors show potential for gene cloning by complementation and co-expression studies.