Construction and dynamic characterization of a Tetrahymena thermophila macronuclear artificial chromosome.
Gene
; 748: 144697, 2020 Jul 20.
Article
en En
| MEDLINE
| ID: mdl-32325092
ABSTRACT
Artificial chromosomes were previously generated for use in bacteria, protists, yeast and human cells. A Tetrahymena thermophila artificial chromosome could serve as a versatile platform to study diverse aspects of Tetrahymena biology and beyond. Here, we placed a C3-type rDNA replication origin and telomere sequences from T. thermophila into a pNeo4 vector, producing the first T. thermophila macronuclear artificial chromosome (TtAC1). Circular or linear forms of TtAC1 can be stably transformed into both vegetative and conjugative T. thermophila cells. Linear TtAC1 was stably double in copy number under antibiotic selection, but its copy number was dropping without antibiotic selection pressure. Southern blot, Real-Time PCR and E. coli retransformation analyses together showed that TtAC1 vector did not integrate into the macronuclear genome, and was maintained as a linear or a circular chromosome in T. thermophila macronucleus under antibiotic selection. The use of TtAC1 for recombinant protein production was demonstrated by western blot analysis of a secreted 27â¯kDa TtsfGFP-12XHis protein. We present the first macronuclear artificial chromosome with species-specific chromosomal elements for use in T. thermophila studies and to aid broad recombinant biotechnology applications.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tetrahymena thermophila
/
Cromosomas Artificiales
Límite:
Animals
Idioma:
En
Revista:
Gene
Año:
2020
Tipo del documento:
Article
País de afiliación:
Turquía