High-efficiency transfection of Acanthamoeba castellanii using a cationic polymer
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae are directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila ) but also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use of A. castellanii and related species as model organisms. Transfecting A. castellanii with plasmids is possible with commercial kits, but it is expensive, inefficient, and vulnerable to product discontinuation. In this contribution, we present a method for efficient transfection of A. castellanii with readily available and inexpensive polyethylenimines. We systematically explore the method’s parameters, obtaining up to 100-fold higher efficiency than currently used protocols. The method presented here provides a robust step towards a complete genetic toolbox for A. castellanii , hence expanding its use as a model organism.
Article activity feed
-
-
General comments: 1-This is a really important work because in this day and age, transforming DNA into an organism or a cell is an essential tool for any molecular biologist
2-And we still don’t understand how to transform the vast majority of organisms on Earth!
3-I applaud this effort for developing robust and accessible transformation tools for the amoeba Acanthamoeba castellanii. I believe this is an important organism to study but equally important are the general trends/approaches about what works to transform an organism
4-accumulating more and more of this knowledge on transformation across different organisms is essential if we want to access the biology of many more organisms
5-Also kudos for the detailed and meticulous transfection optimization! I really enjoyed your use of the N/P ratio and the combinatorial approach over a …
General comments: 1-This is a really important work because in this day and age, transforming DNA into an organism or a cell is an essential tool for any molecular biologist
2-And we still don’t understand how to transform the vast majority of organisms on Earth!
3-I applaud this effort for developing robust and accessible transformation tools for the amoeba Acanthamoeba castellanii. I believe this is an important organism to study but equally important are the general trends/approaches about what works to transform an organism
4-accumulating more and more of this knowledge on transformation across different organisms is essential if we want to access the biology of many more organisms
5-Also kudos for the detailed and meticulous transfection optimization! I really enjoyed your use of the N/P ratio and the combinatorial approach over a range of DNA and PEI concentrations. This is solid science!
Specific comments: 1-Figure 2 → Is it possible to include cell counts, in addition to the RFU signal? This is not a major comment. It’s just that there are cell counts in your other figures so it might be good to include for this figure as well. No need to repeat this experiment if you don’t have cell counts though!
2-Figure 4 → Would it be possible to include arrows or something to indicate which parts of the figure you would like the reader to focus on? It’s great having all of the data included but it may help your narrative if you point in the figure itself to certain key differences or features of the data. Or you might consider including a table to summarize all the data from the figure? May be the table could contain standard deviation around the average for each treatment (or something to show the distribution of the signal but with a single number)?
3-Figure 6 → Would it be possible to include indication of statistical significance?
Questions: 1-How long are plasmids maintained in the transformed cells?
2-Is there a robust selection that could enable you to produce stably transformed lines?
3-And related to Q2, is it possible to produce stable mutant lines (gene deletions or gene introductions), perhaps by transiently transforming CRISPR genes?
4-Does Acanthamoeba castellanii easily undergo transfection in nature? Is there any evidence for that based on its genome?
5-Related to Q4, are there known viruses that infect Acanthamoeba castellanii? Knowledge about these viruses may inform alternative methods of gene delivery and also serve as evidence for the transfection rate in nature.
6-I was wondering if you have tried transfecting Acanthamoeba castellanii with one big plasmid containing two fluorescent genes? Does that work? If yes, is the gene expression worse or better compared to having the genes on two plasmids and transfecting the plasmids together?
-
General comments: 1-This is a really important work because in this day and age, transforming DNA into an organism or a cell is an essential tool for any molecular biologist
2-And we still don’t understand how to transform the vast majority of organisms on Earth!
3-I applaud this effort for developing robust and accessible transformation tools for the amoeba Acanthamoeba castellanii. I believe this is an important organism to study but equally important are the general trends/approaches about what works to transform an organism
4-accumulating more and more of this knowledge on transformation across different organisms is essential if we want to access the biology of many more organisms
5-Also kudos for the detailed and meticulous transfection optimization! I really enjoyed your use of the N/P ratio and the combinatorial approach over a …
General comments: 1-This is a really important work because in this day and age, transforming DNA into an organism or a cell is an essential tool for any molecular biologist
2-And we still don’t understand how to transform the vast majority of organisms on Earth!
3-I applaud this effort for developing robust and accessible transformation tools for the amoeba Acanthamoeba castellanii. I believe this is an important organism to study but equally important are the general trends/approaches about what works to transform an organism
4-accumulating more and more of this knowledge on transformation across different organisms is essential if we want to access the biology of many more organisms
5-Also kudos for the detailed and meticulous transfection optimization! I really enjoyed your use of the N/P ratio and the combinatorial approach over a range of DNA and PEI concentrations. This is solid science!
Specific comments: 1-Figure 2 → Is it possible to include cell counts, in addition to the RFU signal? This is not a major comment. It’s just that there are cell counts in your other figures so it might be good to include for this figure as well. No need to repeat this experiment if you don’t have cell counts though!
2-Figure 4 → Would it be possible to include arrows or something to indicate which parts of the figure you would like the reader to focus on? It’s great having all of the data included but it may help your narrative if you point in the figure itself to certain key differences or features of the data. Or you might consider including a table to summarize all the data from the figure? May be the table could contain standard deviation around the average for each treatment (or something to show the distribution of the signal but with a single number)?
3-Figure 6 → Would it be possible to include indication of statistical significance?
Questions: 1-How long are plasmids maintained in the transformed cells?
2-Is there a robust selection that could enable you to produce stably transformed lines?
3-And related to Q2, is it possible to produce stable mutant lines (gene deletions or gene introductions), perhaps by transiently transforming CRISPR genes?
4-Does Acanthamoeba castellanii easily undergo transfection in nature? Is there any evidence for that based on its genome?
5-Related to Q4, are there known viruses that infect Acanthamoeba castellanii? Knowledge about these viruses may inform alternative methods of gene delivery and also serve as evidence for the transfection rate in nature.
6-I was wondering if you have tried transfecting Acanthamoeba castellanii with one big plasmid containing two fluorescent genes? Does that work? If yes, is the gene expression worse or better compared to having the genes on two plasmids and transfecting the plasmids together?
-