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
Read preprint
(No Ratings Yet)Sign up to customise the site to your preferences and to receive alerts
Register hereAlso in the cell biology category:
Motor Clustering Enhances Kinesin-driven Vesicle Transport
Sharvari Pitke
Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis
Ankita Walvekar
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Also in the developmental biology category:
Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis
Ankita Walvekar
Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation
Ryan Harrison
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos
Anchel De Jaime Soguero
Also in the genetics category:
Intracellular diffusion in the cytoplasm increases with cell size in fission yeast
Leeba Ann Chacko, Sameer Thukral
Significantly reduced, but balanced, rates of mitochondrial fission and fusion are sufficient to maintain the integrity of yeast mitochondrial DNA
Leeba Ann Chacko
The bat Influenza A virus subtype H18N11 induces nanoscale MHCII clustering upon host cell attachment
Mitchell Sarmie, Mohammed A. Jalloh
preLists in the cell biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
| List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
| List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
| List by | Dey Lab, Amanda Haage |
Also in the developmental biology category:
BSDB/GenSoc Spring Meeting 2024
A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.
| List by | Joyce Yu, Katherine Brown |
GfE/ DSDB meeting 2024
This preList highlights the preprints discussed at the 2024 joint German and Dutch developmental biology societies meeting that took place in March 2024 in Osnabrück, Germany.
| List by | Joyce Yu |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
Single Cell Biology 2020
A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.
| List by | Alex Eve |
Society for Developmental Biology 79th Annual Meeting
Preprints at SDB 2020
| List by | Irepan Salvador-Martinez, Martin Estermann |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EDBC Alicante 2019
Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.
| List by | Sergio Menchero et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
SDB 78th Annual Meeting 2019
A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.
| List by | Alex Eve |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Young Embryologist Network Conference 2019
Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London
| List by | Alex Eve |
Pattern formation during development
The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.
| List by | Alexa Sadier |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
| List by | Shikha Nayar |
Also in the genetics category:
BSDB/GenSoc Spring Meeting 2024
A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.
| List by | Joyce Yu, Katherine Brown |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
| List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
| List by | Nándor Lipták |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |