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Stable transformation of Trypanosoma brucei
A L ten Asbroek1, C A Mol, R Kieft
1Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
Molecular and Biochemical Parasitology
|May 1, 1993
Summary
Stable transformation of Trypanosoma brucei is most efficient using linear DNA, which integrates into chromosomes via homologous recombination. Gene amplification occurs through unequal sister chromatid exchange, with extrachromosomal replication being rare.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Trypanosoma brucei is a significant protozoan parasite.
- Stable transformation is crucial for genetic manipulation of T. brucei.
- Understanding DNA integration mechanisms is key for effective gene targeting.
Purpose of the Study:
- To investigate factors influencing stable transformation in Trypanosoma brucei.
- To elucidate the mechanisms of exogenous DNA integration and gene amplification.
- To assess the potential for extrachromosomal DNA replication in T. brucei.
Main Methods:
- Analysis of transformation efficiency using linear versus circular DNA constructs.
- Investigation of homologous recombination in DNA integration.
- Selection for increased drug resistance to study gene amplification.
- Attempted generation of extrachromosomal replicating vectors.
Main Results:
- Linear DNA demonstrated higher transformation efficiency than circular DNA.
- Homologous recombination was the predominant integration mechanism.
- Non-homologous DNA flanking sequences reduced transformation efficiency.
- Neomycin phosphotransferase (neo(r)) gene amplification occurred via unequal sister chromatid exchange.
- Extrachromosomal plasmid replication was largely unsuccessful, with one instance of a pentameric construct.
Conclusions:
- Trypanosoma brucei exhibits a strong preference for chromosomal integration of exogenous DNA through homologous recombination.
- Unequal sister chromatid exchange is a likely mechanism for gene amplification in T. brucei.
- Developing stable extrachromosomal vectors in T. brucei remains challenging.