Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery
Posted on: 9 May 2024
Preprint posted on 14 April 2024
Categories: cell biology, developmental biology, genetics
Background:
Delivering DNA or RNA is an important strategy for uncovering gene function that has been widely applied to animal models, including zebrafish. Currently, microinjections are the gold standard technique for nucleic acid delivery into zebrafish embryos. While it is highly efficient, it can be technically challenging, and requires substantial training for new-commers in a research laboratory. For example, most experiments require genetic manipulation to happen at the single-cell stage, with a very narrow time window left to collect embryos, load needles for injection, and perform the injection, creating a major barrier towards achieving high-throughput generation of genetically modified lines. Further, microinjections require specialized equipment that may not be accessible to all laboratories.
There has been a large focus over the last decades on improving methods for mutagenesis and transgenesis, with some efforts aimed towards improving the technical aspects of genetic material delivery, including the development of techniques like MIC-drop (Parvez et al. 2021) and electroporation (Kumar et al., 2019). In this preprint, the authors offer a new alternative method that bypasses the laborious, time-consuming layers that come with microinjections in zebrafish. They propose, and test, lipid-based transfection as a cost-effective, simple and high-throughput genetic delivery method.
Figure 1. Lipofection can be used to generate Tol2 transgenic lines in zebrafish without microinjections. Schematic representation of multiple constructs transfected in parallel with the plasmid transfection reagent Lipofectamine LTX.
Key findings:
mRNA can be effectively delivered into zebrafish embryos with lipid-based transfection using a consistent and reproducible method
In this preprint, the authors tested a novel lipofection protocol in zebrafish by exposing single-cell stage zebrafish embryos to StayGold mRNA, which codes for a highly stable green fluorescent protein derived from jellyfish (Hirano et al., 2022), and a liposome carrier molecule (lipofectamine). This method is fairly simple and fast, requiring removal of the chorion with an enzyme, and incubating de-chorionated embryos with a mixture of a liposome carrier molecule (lipofectamine) with the DNA/RNA material to be delivered. After incubating single-cell stage zebrafish embryos with lipofectamine and StayGold mRNA, the authors observed strong fluorescence in 95% of treated embryos, with no signs of toxicity, normal development, and no differences in survival in comparison with untreated controls. This suggested mRNA lipid-based transfection is highly efficient in zebrafish. The authors also measured fluorescence intensity and quantified mRNA expression on embryos that were transfected with several concentrations of of StayGold mRNA. They found that fluorescence intensity and mRNA quantity were consistent with the amount of mRNA used for transfection, suggesting consistent and reproducible results with this method.
Transfection allows for flexible incubation times and sparse labeling of embryos
While the authors used an incubation time for transfection of 4 hours, they also noted that 1 hour was enough to ensure efficient transfection in zebrafish embryos, suggesting a flexible incubation time-window. Remarkably, mRNA delivery through lipofection was also successful later in development, including at the 2-, 4-, 8- and 16-cell stages. This is relevant for studies requiring mosaic or sparse fluorescence for imaging.
Multiple Tol2 plasmids can be effectively delivered into zebrafish embryos in parallel with lipid-based transfection
To determine if this method could be used for generating transgenic lines with reporter gene expression, the authors co-transfected Tol2 plasmids and Tol2 mRNA with their lipid-based transfection method. They evaluated fluorescence expression from 1 day to 7 days post transfection and found an 80% efficiency in transgenesis. Remarkably, they showed that multiple constructs can be delivered at once through this method in a cost-effective and rapid way.
What I like about the preprint/Why this work is important:
While microinjections are an accessible strategy for delivering DNA/RNA material into zebrafish embryos, the technique requires multiple steps that precede delivery of genetic material into the cell. The method proposed in this preprint eliminates most of these steps and enables genetic modification with minimal laboratory personnel training. This cost-effective method has the potential to accelerate the rate at which new transgenic and mutant lines are made in most zebrafish laboratories with a molecular biology setup.
References:
- Parvez, S., Herdman, C., Beerens, M., Chakraborti, K., Harmer, Z. P., Yeh, J. J., MacRae, C. A., Yost, H. J., & Peterson, R. T. (2021). MIC-Drop: A platform for large-scale in vivo CRISPR screens. Science (New York, N.Y.), 373(6559), 1146–1151. https://doi.org/10.1126/science.abi8870
- Kumar, P., Nagarajan, A., & Uchil, P. D. (2019). Electroporation. Cold Spring Harbor protocols, 2019(7), 10.1101/pdb.top096271. https://doi.org/10.1101/pdb.top096271
- Hirano, M., Ando, R., Shimozono, S. et al.A highly photostable and bright green fluorescent protein. Nat Biotechnol 40, 1132–1142 (2022). https://doi.org/10.1038/s41587-022-01278-2
doi: https://doi.org/10.1242/prelights.37323
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