Name: fast and efficient expression of multiple proteins in avian embryos using mrna electroporation
Uploaded: 2019-06-07T14:30:00
Description: Watch this Scientific Journal Video about Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation at JoVE.com

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&#8217;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.

All animal procedures were carried out in accordance with approved guidelines from the Children&#8217;s Hospital Los Angeles and the University of Southern California Institutional Animal Care and Use Committees.
1. Generation pCS2-based Expression Vectors
<ol>
	<li>To clone pCS2.Lifeact-eGFP, prepare the vector backbone by digesting 2 &#181;g 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...

<ol>
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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...

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 cells4,5,6,7. However, several experimental limitations also exist with DNA electroporation8. For instance, DNA electroporation often introduces highly variable numbers of expression vectors per cell and subsequently the mRNAs and proteins they encode. This variability can lead to considerable cell-cell heterogeneity that complicates both image analysis and data interpretation9,10. Additionally, proteins from DNA electroporation only begin to express ~3 h post-electroporation and do not reach the maximum efficiency in cell number and fluorescence intensity until 12 h, likely due to the time required to transfer into the nucleus and complete both transcription and translation in vivo11.
In contrast, mRNA transfection has been effectively used in a variety of model systems, including Xenopus laevis oocytes by microinjection12,13, reprogramming human stem cells by mRNA lipofectamine transfection14, and electroporating recalcitrant neural stem cells in adult mice15. We tested the ability of mRNA electroporation to efficiently label cells during early avian embryonic development using in vitro synthesized mRNAs that encode distinct fluorescent proteins (FPs). For our studies, we used the pCS2+ vector, a multipurpose expression vector that is commonly used for expressing proteins in Xenopus and zebrafish embryos. The SP6 and T7 RNA polymerase promoters in the pCS2+ permit the synthesis of mRNA and protein&#160;from any cloned gene when used in an&#160;in vitro&#160;transcription/translation system.
Here, we demonstrate that mRNA electroporation allows fast and efficient expression of fluorescent proteins (FPs) in gastrulating quail embryos. We designed and generated many of the expression vectors used in these studies. For example, we subcloned the LifeAct-eGFP gene16 into the pCS2+ vector17 to express from the CMV promoter and SP6 promoter. The inserted gene lies downstream of the SP6 promoter and upstream of the SV40 poly(A) tail18. In embryos co-electroporated with mRNA and DNA, FPs encoded from in vitro transcribed mRNAs were first detected within 20 min of electroporation, whereas FPs from DNA expression vectors were detected only after 3 h. Multiple mRNAs encoding for nuclear, Golgi, and membrane proteins can be electroporated into an embryo simultaneously, resulting in the quick and efficient expression of multiple proteins in individual cells. Finally, using an in vivo fluorescence recovery after photobleaching (FRAP) assay, we show that a majority of the electroporated mRNAs decay within 2 h. Thus, fast initial protein production combined with limited new protein translation makes mRNA electroporation a valuable technique when temporal control of expression is necessary.

<table>
	<tbody>
		<tr>
			<td>BamHI-HF</td>
			<td>New England Biolabs</td>
			<td>R3136L</td>
			<td></td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England Biolabs</td>
			<td>R0144S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsrG1-HF</td>
			<td>New England Biolabs</td>
			<td>R3575S</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI-HF</td>
			<td>New England Biolabs</td>
			<td>R3189L</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI-HF</td>
			<td>New England Biolabs</td>
			<td>R3138L</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol</td>
			<td>Thermo Fisher</td>
			<td>15593031</td>
			<td></td>
		</tr>
		<tr>
			<td>SP6 mMessage Machine in vitro transcription kit</td>
			<td>Thermo Fisher</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma Aldrich</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>93443</td>
			<td>4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td>2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride, DAPI dihydrochloride</td>
		</tr>
		<tr>
			<td>Whatman No.1 filter paper</td>
			<td>Sigma Aldrich</td>
			<td>WHA1001125</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>51457</td>
			<td></td>
		</tr>
		<tr>
			<td>pmTurquoise2-Golgi</td>
			<td>Addgene</td>
			<td>36205</td>
			<td>pmTurquoise2-Golgi was a gift from Dorus Gadella (Addgene plasmid # 36205 ; http://n2t.net/addgene:36205 ; RRID:Addgene_36205)</td>
		</tr>
		<tr>
			<td>pmEGFP-N1-LifeAct</td>
			<td></td>
			<td></td>
			<td>Nat. Methods 2008;5:605-7. PubMed ID: 18536722</td>
		</tr>
		<tr>
			<td>pCS2.Lifeact-mGFP</td>
			<td>Addgene</td>
			<td></td>
			<td>This paper</td>
		</tr>
		<tr>
			<td>pCS.H2B-citrine</td>
			<td>Addgene</td>
			<td>53752</td>
			<td>pCS-H2B-citrine was a gift from Sean Megason (Addgene plasmid # 53752 ; http://n2t.net/addgene:53752 ; RRID:Addgene_53752)</td>
		</tr>
		<tr>
			<td>pCS.memb-mCherry</td>
			<td>Addgene</td>
			<td>#53750</td>
			<td>pCS-memb-mCherry was a gift from Sean Megason (Addgene plasmid # 53750 ; http://n2t.net/addgene:53750 ; RRID:Addgene_53750)</td>
		</tr>
		<tr>
			<td>Zeiss LSM-780 inverted microscope</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td></td>
			<td>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 (&#62;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.</td>
		</tr>
		<tr>
			<td>CUY-21 EDIT in vivo electroporator</td>
			<td>Bex Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum flat square electrode, side length 5 mm</td>
			<td>Bex Co., Ltd.</td>
			<td>LF701P5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus MVX10 FL Stereo Microscope</td>
			<td>Olympus LifeScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XM10 Monochrome camera</td>
			<td>Olympus LifeScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) for HCR (10&#215;, pH 7.4) </td>
			<td></td>
			<td></td>
			<td>To prepare 1 L of a 10&#215; 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).</td>
		</tr>
		<tr>
			<td>1.37 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27 mM KCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>80 mM Na2HPO4 20 mM KH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS/Triton</td>
			<td></td>
			<td></td>
			<td>Add 1 mL of Triton X-100 (Sigma Aldrich 93443) and 100 mL of 10&#215; PBS to 890 mL of ultrapure distilled H2O. Filter the solution through a 0.2-&#956;m filter and store it at 4 &#778;C until use.</td>
		</tr>
		<tr>
			<td>1&#215; phosphate-buffered saline (PBS) (DEPC-treated; pH 7.4)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1% Triton X-100</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fast and efficient expression of multiple proteins in avian embryos using mrna 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.

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 &#181;g/&#181;L, we used an equimolar concentration of mRNA (calculated to be around 0.25-0.5 &#181;g/&#181;L for H2B-Citrine) for mRNA electroporation. We first tested the electroporation efficiency of pCS2+.H2B-Citrine DNA compared...

Watch this Scientific Journal Video about Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation at JoVE.com

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

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. ...

mRNA electroporation provides a fast, efficient, and spatially and temporally controlled expression of multiple proteins within the avian model system. This method allows for a more broad and quick expression of fluorescent proteins than the labeling achieved by standard DNA electroporation. Electroporation facilitates the transient opening of pores in the plasma membrane that serve as conduits for the transfer of genetic payloads like RNA and DNA into the cells of numerous model organisms.For the initial experiments work with embryos that are older than a day old since they are hardier and more resistant to external manipulations like electroporation. At the appropriate embryonic developmental stage gently break and pour the quail egg contents into a 10 centimeter Petri dish. Use a transfer pipette to remove the majority of the thick albumen.Use a lab tissue to gently wipe the surface of the yoke to remove the remaining thick albumen around the embryo to ensure that the embryo will stick tightly to the paper ring. Lay precut filter paper over the embryo and use scissors to smoothly cut around the perimeter of the embryo. Use a Pasteur pipette to layer PPS under the embryo using gentle streams to vacate any yoke sticking to the tissue.Slowly pull the embryo paper ring at an oblique angle up and off of the yoke. Place the paper into a Petri dish filled with PBS for further cleaning. Once most of the yoke has been removed, place the embryo ventral side up in a 35 millimeter Petri dish covered with a semisolid mixture of agar and albumen.Prepare the electroporation chamber by connecting the black electrode filling it with PBS and placing the embryo inside. Use a glass microcapillary to inject a bolus of 200 nanoliters of mRNA into the cavity between the epiblast and vitelline membrane covering the desired region. And then use the red electrode to electroporate the embryo.Place the electroporated embryo back onto the agar-albumen mixture and incubate at 38 degrees Celsius until the desired developmental stage. For imaging of the electroporation-encoded fluorescent proteins observe all of the electroporated embryos under a fluorescent dissecting stereoscope to select the healthiest and best electroporated embryo for the dynamic imaging experiments. Continue to incubate the other electroplated embryos and non-electroplated embryos in a separate incubator.Briefly rinse the selected embryo with PBS to remove any bubbles that may have formed on the dorsal side of the embryo during the electroporation. Place the cleaned embryo directly into an imaging dish containing a thin layer of about 150 microliters of albumen-agar taking care not to generate any bubbles on the dorsal surface of the embryo. Add a small moist rolled up piece of tissue paper along the inside edges of the imaging dish.Seal the dish with paraffin film to minimize evaporation during the imaging and incubation. Move the dish quickly to the prewarmed stage of a confocal microscope and use the brightfield channel to locate the colored dye in the embryo. Set the imaging software to the desired objective, dichroic mirror, and emission spectra, and turn on an appropriate laser.Take care that the cells will remain in the image vision for the entirety of the movie. Use a high enough imaging resolution, and ensure that the imaging conditions will not cause phototoxicity. Click Live in the imaging software and adjust the laser power to a setting that is appropriate for the fluorescence intensity depending on each microscope laser power.Begin imaging the embryo using 1%laser power and an 800 gain, increasing the laser power slowly by 1%increments until saturated pixels are seen. When saturated pixels are observed decrease the laser power slightly until no saturated pixels can be visualized. Image the embryo every three to five minutes to check the migration of individual cells across different time points using five micrometer Z-stacks of the entire electroplated area with some extra room toward the bottom of the Z-stack in case the embryo sinks into the agarose bed during the imaging session.To observe how fast the cells are moving check the first few time points of the first movie. If the cells are moving at a fast rate, consider expanding the zoom of the imaged area or imaging a different region. When the entire electroplated region of the embryo has been imaged, image in unelectroplated region of the same embryo to determine the autofluorescence levels.To determine how long transfected mRNA can be translated into fluorescent proteins after confirming electroporation of the gene of interest on the stereo microscope, place the embryo on a preheated stage of the inverted confocal microscope and use the 405 nanometer laser with a 70%laser power, 100 iterations, and a scan speed of four to photobleach most of the cellular fluorescence from the electroplated gene at a variety of time points. Continue to incubate the embryos on the stage at 36 degrees Celsius after photo bleaching, taking care to pay attention to the actively dividing cells within the photobleached region that are typically only seen in healthy embryos and thus indicate that the electroporation did not harm the embryonic cells. Acquire post-bleach Z-stack images of the electroplated region for a desired length of time at regular three to five minute time intervals.To ensure that the imaging conditions do not deleteriously affect the embryo survival concurrently incubate electroplated embryos that are not photobleached to serve as a control for the imaging. To quantitate the photobleaching results for the mRNA decay post-electroporation use ImageJ to measure the fluorescence intensity of a 7.5 micrometer circle in the center of the electroplated area of each cell to track the cell fluorescence over each three to five minute interval. When all of the cells within the photobleached region that are not undergoing mitosis and have been completely photobleached have been measured, plot the fluorescence intensity over time post-bleach at each of the time points the embryo was photobleached.Although DNA electroporation leads to a brighter fluorescence in some electroplated cells, the efficiency of DNA electroporation is visibly lower compared to the widely expressed mRNA-encoded fluorescent proteins. When comparing DNA and mRNA co-electroporation within the same embryo, the DNA expressing fluorescent protein efficiency is also significantly and consistently less efficient than that of the mRNA expressing fluorescent protein. Quantification of all of the embryos electroplated with only mRNA, DNA or a combination of the two, reveals that mRNA transfects about 75%of the cells in a given region, while DNA only transfects about 25%of the cells.The transfected mRNA should lead to a faster protein production compared to transfected DNA since the mRNA can be immediately recognized by cytosolic translation machinery. Although the co-electroporation of multiple DNAs has previously been shown to be relatively inefficient, the co-electroporation of four mRNAs in this experiment resulted in an 87%transfection efficiency of all four of the mRNAs within all of the electroplated regions. Although photobleaching initially reduces the fluorescence intensity in the photobleached cells by about 95%some cells remain that recover their ability to divide and express fluorescent proteins shortly after electroporation.This ability decreases over five hours post-electroporation however. Take your time optimizing the best imaging conditions and continue to tinker even after starting the movie being ready to make any adjustments if needed.

fast-efficient-expression-multiple-proteins-avian-embryos-using-mrna

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Tracing Gene Expression Through Detection of &amp;#946;-galactosidase Activity in Whole Mouse Embryos

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.