Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

Summary

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.

Abstract

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.

Introduction

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 cells^1,2,3. 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 cells^4,5,

Protocol

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

1. To clone pCS2.Lifeact-eGFP, prepare the vector backbone by digesting 2 µg of pCS2.CycB1-GFP (a construct containing a different insert) with BamHI (10 U) and BsrGI (10 U) in appropriate digestion buffer (see Table of.......

Discussion

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.......

Materials

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