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We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.
We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.
Electroporation is a physical transfection method that uses an electrical pulse to create transient pores in the plasma membrane, allowing substances like nucleic acids or chemicals to pass into the cytosol. Electroporation is widely used to deliver DNA into bacteria, yeast, plants, and mammalian cells1,2,3. It is routinely used to introduce genetic payloads into target cells and tissues within the developing avian embryo to study the genetic control of development or label migrating populations of cells4,5,
All animal procedures were carried out in accordance with approved guidelines from the Children’s Hospital Los Angeles and the University of Southern California Institutional Animal Care and Use Committees.
1. Generation pCS2-based Expression Vectors
mRNA electroporation is more efficient than DNA electroporation
We used pCS2+.H2B-Citrine to prepare in vitro transcribed mRNA. Since DNA electroporation is usually performed at 1-2 µg/µL, we used an equimolar concentration of mRNA (calculated to be around 0.25-0.5 µg/µL for H2B-Citrine) for mRNA electroporation. We first tested the electroporation efficiency of pCS2+.H2B-Citrine DNA compared.......
In this protocol, we provided step by step instructions on how to precisely microinject and electroporate mRNA into the cells of gastrulating quail embryos. We demonstrated that in vitro synthesized mRNA electroporation allows fast and efficient expression of fluorescent proteins (FPs) in gastrulating quail embryos (Figure 2 and 3). Fluorescence from H2B-citrine protein translated from electroporated mRNAs could be detected by confocal microscopy within ~20 min and increased.......
We thank David Huss for helpful insights into this work. This work was supported in part by the Rose Hills Foundation Summer Research Fellowship (2016-2018) and USC Provost’s Undergrad Research Fellowship to M.T., the Saban Research Institute Intramural Training Pre-Doctoral Award to M.D., and the University of Southern California Undergraduate Research Associates Program award to R.L.
....Name | Company | Catalog Number | Comments |
BamHI-HF | New England Biolabs | R3136L | |
BglII | New England Biolabs | R0144S | |
BsrG1-HF | New England Biolabs | R3575S | |
NotI-HF | New England Biolabs | R3189L | |
SalI-HF | New England Biolabs | R3138L | |
Phenol:Chloroform:Isoamyl Alcohol | Thermo Fisher | 15593031 | |
SP6 mMessage Machine in vitro transcription kit | Thermo Fisher | AM1340 | |
Fast Green FCF | Sigma Aldrich | F7252 | |
Triton X-100 | Sigma Aldrich | 93443 | 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether |
DAPI | Sigma Aldrich | D9542 | 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4′,6-Diamidino-2-phenylindole dihydrochloride, DAPI dihydrochloride |
Whatman No.1 filter paper | Sigma Aldrich | WHA1001125 | |
glycerol | Sigma Aldrich | G9012 | |
Urea | Sigma Aldrich | 51457 | |
pmTurquoise2-Golgi | Addgene | 36205 | pmTurquoise2-Golgi was a gift from Dorus Gadella (Addgene plasmid # 36205 ; http://n2t.net/addgene:36205 ; RRID:Addgene_36205) |
pmEGFP-N1-LifeAct | Nat. Methods 2008;5:605-7. PubMed ID: 18536722 | ||
pCS2.Lifeact-mGFP | Addgene | This paper | |
pCS.H2B-citrine | Addgene | 53752 | pCS-H2B-citrine was a gift from Sean Megason (Addgene plasmid # 53752 ; http://n2t.net/addgene:53752 ; RRID:Addgene_53752) |
pCS.memb-mCherry | Addgene | #53750 | pCS-memb-mCherry was a gift from Sean Megason (Addgene plasmid # 53750 ; http://n2t.net/addgene:53750 ; RRID:Addgene_53750) |
Zeiss LSM-780 inverted microscope | Carl Zeiss Microscopy GmbH | The LSM-780 is a confocal and multi-photon microscope that offers the sensitivity required for vital imaging work. Equipped with a motorized stage, an autofocus device, and a full stage-top blackout incubator, the 780 is an excellent microscope for high-end live cell/embryo imaging. The high-sensitivity 32-channel Quasar detector allows for spectral imaging, linear unmixing, and high color count (>4) image acquisition. Excitation can be performed with 6 lines single photon lasers (405, 458, 488, 514, 564 and 633 nm), Chameleon (Coherent) 2-photon laser (range from 690nm to 1000nm), and run with ZEN 2011 SP7 (Black) system software. | |
CUY-21 EDIT in vivo electroporator | Bex Co., Ltd. | ||
Platinum flat square electrode, side length 5 mm | Bex Co., Ltd. | LF701P5E | |
Olympus MVX10 FL Stereo Microscope | Olympus LifeScience | ||
XM10 Monochrome camera | Olympus LifeScience | ||
Phosphate-Buffered Saline (PBS) for HCR (10×, pH 7.4) | To prepare 1 L of a 10× stock solution, combine 80 g of NaCl (Sigma-Aldrich S3014), 2 g of KCl (Sigma-Aldrich P9541), 11.4 g of Na2HPO4 (anhydrous; Sigma-Aldrich S3264), and 2.7 g of KH2PO4 (anhydrous; Sigma-Aldrich P9791). Adjust the pH to 7.4 with HCl, and bring the final volume to 1 L with ultrapure H2O. Avoid using CaCl2 and MgCl2 in PBS for HCR. It is important that the PBS for HCR is prepared as an RNase-free solution (e.g., via diethylpyrocarbonate [DEPC] treatment). | ||
1.37 M NaCl | |||
27 mM KCl | |||
80 mM Na2HPO4 20 mM KH2PO4 | |||
PBS/Triton | Add 1 mL of Triton X-100 (Sigma Aldrich 93443) and 100 mL of 10× PBS to 890 mL of ultrapure distilled H2O. Filter the solution through a 0.2-μm filter and store it at 4 ̊C until use. | ||
1× phosphate-buffered saline (PBS) (DEPC-treated; pH 7.4) | |||
0.1% Triton X-100 |
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