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Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata
Abstract:
Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences. Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization. Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell. The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA. Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome. The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions.
Insights
Researchers introduced a plasmid into Crithidia fasciculata cells, creating genetically modified organisms resistant to paromomycin. The introduced plasmid DNA was unstable and often lost within 30 cell divisions.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Crithidia fasciculata is a protozoan parasite relevant in biological research.
- Genetic manipulation of protozoan parasites is crucial for understanding their biology and developing control strategies.
Purpose of the Study:
- To establish a method for introducing and maintaining foreign DNA in Crithidia fasciculata.
- To investigate the stability and characteristics of extrachromosomal plasmids in this organism.
Main Methods:
- Transfection of Crithidia fasciculata with a plasmid (pDK96) containing antibiotic resistance genes and replication sequences.
- Selection of resistant colonies using paromomycin.
- Plasmid recovery and characterization via hybridization and transformation of E. coli.
Main Results:
- Approximately 1% of resistant colonies contained extrachromosomal plasmids.
- Recovered plasmids were often deleted, lacking the autonomous replication sequence.
- A unique fragment of Crithidia nuclear DNA was identified in some recovered plasmids.
- Plasmid DNA exhibited instability, being lost within 30 cell divisions even under selection.
Conclusions:
- Successful, albeit inefficient, introduction of foreign DNA into Crithidia fasciculata is achievable.
- Extrachromosomal plasmids in Crithidia fasciculata are inherently unstable.
- Further optimization is needed for stable genetic modification of this parasite.