Name: in utero electroporation of multiaddressable genome-integrating color (magic) markers to individualize cortical mouse astrocytes
Uploaded: 2020-05-21T15:00:00
Description: Watch this Scientific Journal Video about In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes at JoVE.com

We thank S. Fouquet and the imaging and animal core facilities of Institut de la Vision and Institut des Neurosciences de Montpellier (MRI and RAM) for technical assistance. This work was supported by fellowships from R&#233;gion Ile-de-France and Fondation ARC pour la Recherche sur le Cancer to S.C, and from Universit&#233; Paris-Saclay (Initiatives Doctorales Interdisciplinaires) to L.A., by funding from European Research Council (ERC-SG 336331, PI J. Valette) to E.H., by Agence Nationale de la Recherche under contracts ANR-10-LABX-65 (LabEx LifeSenses), ANR-11-EQPX-0029 (Equipex Morphoscope2), ANR-10-INBS-04 (France BioImaging), by Fondation pour la Recherche M&#233;dicale (Ref. DBI20141231328), by the European Research Council (ERC-CoG 649117, PI J. Livet) and by ATIP-Avenir program (PI K. Loulier).

All animal procedures described here were carried out in accordance with institutional guidelines. Animal protocols have been approved by the Charles Darwin animal experimentation ethical board (CEEACD/N&#176;5).
1. Preparation of endotoxin-free plasmids for MAGIC Markers in utero electroporation
<ol>
	<li>Bacterial transformation
	<ol>
		<li>On ice, thaw DH5 alpha competent cells stored at -70 &#176;C.</li>
		<li>Warm up the agar plates containing the appropriate antibiotic (100 &#181;g/mL ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nature Biotechnology. 28 (4), 348-353 (2010).</li><li>Peng, H., Bria, A., Zhou, Z., Iannello, G., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensible+visualization+and+analysis+for+multidimensional+images+using+Vaa3D.">Extensible visualization and analysis for multidimensional images using Vaa3D.</a> Nature Protocols. 9 (1), 193-208 (2014).</li><li>Peng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+finger+boosts+three-dimensional+imaging+and+microsurgery+as+well+as+terabyte+volume+image+visualization+and+analysis.">Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis.</a> Nature Communications. 5 (1), 4342 (2014).</li><li>Loulier, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Cell+and+Lineage+Tracking+with+Combinatorial+Labels.">Multiplex Cell and Lineage Tracking with Combinatorial Labels.</a> Neuron. 81 (3), 505-520 (2014).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108 (2), 193-199 (1991).</li><li>Abdeladim, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+multiscale+brain+imaging+with+chromatic+multiphoton+serial+microscopy.">Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.</a> Nature Communications. 10 (1), 1662 (2019).</li><li>Chen, F., LoTurco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+stable+transgenesis+of+radial+glia+lineage+in+rat+neocortex+by+piggyBac+mediated+transposition.">A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.</a> Journal of Neuroscience Methods. 207 (2), 172-180 (2012).</li><li>Siddiqi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+Mapping+by+PiggyBac+Transposase+Reveals+That+Neocortical+GLAST++Progenitors+Generate+More+Astrocytes+Than+Nestin++Progenitors+in+Rat+Neocortex.">Fate Mapping by PiggyBac Transposase Reveals That Neocortical GLAST+ Progenitors Generate More Astrocytes Than Nestin+ Progenitors in Rat Neocortex.</a> Cerebral Cortex. 24 (2), 508-520 (2014).</li><li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes to Cells. 15 (5), 501-502 (2010).</li><li>LoTurco, J., Manent, J. 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In utero electroporation (IUE) of MAGIC Markers in cortical progenitors (Figure 1) enabled labeling of astrocytes throughout the postnatal cerebral cortex at different postnatal stages (P4-P7-P21, Figure 2). Interestingly, the stage of IUE is not critical, as electroporation performed from E13.5 to E15.5 yields similar labeling patterns concerning cortical astrocytes14. However, the location of labeled pyramidal neurons in the cortical pa...

Astrocytes play numerous vital functions in brain development and physiology1. Beside their role at the blood-brain barrier where they regulate nutrient uptake and blood flow, they actively contribute to synapse formation and function while producing neuromodulators that can alter neuronal activity and behavior2. Furthermore, astrocyte dysfunction contributes to a variety of neurological disorders3. Astrocytes located in the cerebral cortex display an elaborate morphology enabling extensive contact with neuronal processes. These contacts, essential for circuit function, also control astrocyte morphogenesis and synaptogenesis through cell-adhesion proteins4. Neuroscientists need convenient and robust tools to investigate astrocyte development and morphogenesis in their neurological models of interest. However, due to the close apposition of astrocytes&#160;to their&#160;neighbors and their uniform three-dimensional tiling, it is challenging to single out cortical astrocytes and comprehensively assess their morphology using immunomarkers.
Currently, two main genetic engineering strategies enable labeling and individualization of cortical astrocytes in situ: sparse reporter activation in transgenic mouse lines or somatic transgenesis using electroporation of reporter plasmids. The first strategy relies on breeding a floxed reporter mouse line with mice expressing an inducible form of Cre recombinase activated specifically in astrocytes upon tamoxifen delivery (e.g., Aldh1l1-CreERT25). Several disadvantages are associated with this strategy. First, breeding transgenic mice requires a large number of animals and multiple assays are typically needed to determine the proper dose of tamoxifen to provide adequately sparse labeling of cortical astrocytes. Analyzing cortical astrocyte phenotypes in a genetic mouse model of interest will require even more breeding and mouse consumption. Furthermore, in utero tamoxifen injection is known to interfere with parturition, making this strategy difficult to apply to the study of the earliest stages of astrocyte development. In vivo DNA electroporation is an alternative tamoxifen-free strategy that relies on a minimum number of animals6. Performed either at embryonic or postnatal stages, this approach consists of injecting reporter plasmids in the lateral ventricles of rodents followed by electric pulses that create pores in the cell membrane, hence allowing DNA to enter progenitor cells lining the ventricle. Subsequently, the reporter transgenes carried by the electroporated plasmids are processed by the targeted cell machinery and expressed7. Two electroporation methods have been previously described to label mouse cortical astrocytes: 1) Postnatal Astrocyte Labeling by Electroporation (PALE), which relies on the electroporation of 1&#8211;2 single-color episomal reporter plasmids at early postnatal stages4; 2) The StarTrack strategy based on in utero electroporation (IUE) of multiple single-color integrative reporter plasmids8,9,10. Although these two techniques efficiently label PrA in the cerebral cortex, they also present some limitations. In their initial version, both methods rely on a glial fibrillary acidic protein (GFAP) promoter to drive expression in astrocytes, which may bias the labelling toward radial glia as well as pial and reactive astrocytes that express GFAP more strongly than normal resting PrA11,12. Regarding PALE, other disadvantages are the late stage of electroporation, which prevents labeling of early-born PrA (or those originating from early delaminating progenitors) and analysis of early stages of astroglia development, and the use of episomal vectors that become diluted through successive divisions during the massive proliferation that PrA undergo during the first postnatal week13,14. In contrast to PALE, StarTrack is based on the embryonic electroporation of integrative reporter plasmids that allow tracking the contribution of both embryonic and postnatal progenitors to PrA. An updated StarTrack scheme relying on the ubiquitin C promoter (UbC-StarTrack) achieves broader expression of fluorescent reporters in both the neuronal and glial descent (astrocytes included) of neural progenitors15,16,17. However, in its current version, implementation of this approach is complex, as it relies on an equimolar mixture of 12 distinct plasmids expressing six fluorescent proteins (FP) with partial excitation and emission spectra overlap.
Presented here is a straightforward in utero electroporation-based multicolor labeling method using integrative reporter constructs driven by a strong and broadly active promoter to single out cortical astrocytes14. In addition, an easy image analysis pipeline using both licensed (e.g., Imaris) and open access (Vaa3D18,19,20) image analysis software is provided to segment astrocyte territorial volume and arborization, respectively. Compared to the previously described methods, this strategy relies solely on 1&#8211;2 multicolor integrative transgenes Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM21) directed to the cytoplasmic and (optionally) nuclear cell compartment whose expression is driven by a synthetic CAG promoter comprised of a cytomegalovirus enhancer, chicken &#946;-actin promoter, and rabbit &#946;-globin splice acceptor site22. This enables labeling and tracking of cortical astrocytes, from embryonic to late postnatal stages, independent of GFAP expression14,23. Each of these transgenes bears the following four distinct FP: eBFP, mTurquoise2/mCerulean, EYFP, and tdTomato/mCherry, which display minimal spectral overlap that can be easily circumvented with 1) Sequential channel acquisition; 2) Optimized excitation power and collection gain; and 3) Specific dichroic filters to collect narrow FP emission windows. The MM strategy uses Cre/lox recombination with a self-excisable Cre recombinase (seCre) to drive stochastic expression of FP in a cellular population. A single copy of MM transgene expresses FP in a mutually exclusive manner, while multiple transgenes give rise to FP combinations, creating dozens of distinct hues. Genomic integration of the transgenes is driven by the piggyBac (PB) or Tol2 transposition system24,25,26. Therefore, through in utero electroporation, the MM toolkit and the multicolor &#8216;mosaic&#8217; that it generates enable simultaneous marking of multiple adjacent cortical progenitors and the tracking of their glial descent, including cortical astrocytes, over long periods. Color contrasts resulting from the expression of distinct FP permit delineation of the contour of PrA and subsequently extract key information about their territorial volume (using IMARIS) and complex morphology (using Vaa3D). The multicolor strategy presented in detail here is a convenient and robust method that gives quick and easy access to the cortical astrocyte surface and morphology in wild type mice at various developmental stages, and is easily adaptable to investigate astrocyte anatomical features in mouse models of neurological diseases without using transgenic reporter lines.

<table>
	<tbody>
		<tr>
			<td>1.1 Bacteria transformation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Euromedex</td>
			<td>EU0400-C</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5 alpha competent cells</td>
			<td>Fisher Scientitic</td>
			<td>11563117</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice box</td>
			<td>Dutscher</td>
			<td>139959</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td>60615</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Fisher Scientitic</td>
			<td>11563117</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile petri dish- 10 cm</td>
			<td>Thermo Fisher</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR</td>
			<td>462-0556H</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.2 Plasmid culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml culture tube</td>
			<td>Dutscher</td>
			<td>187262</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass erlenmeyer- 2L</td>
			<td>Fisher Scientitic</td>
			<td>11383454</td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td>Sigma</td>
			<td>L3522</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 Plasmid DNA preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi Plus EF</td>
			<td>Macherey-Nagel</td>
			<td>740426.10</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1 Preparation of the solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26 G x 1/2 needle</td>
			<td>Terumo</td>
			<td>8AN2613R1</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 1/2 needle</td>
			<td>Terumo</td>
			<td>8AN3013R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Sigma Aldrich</td>
			<td>F7272</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use polypropylene syringe, 1 mL</td>
			<td>Dutscher</td>
			<td>50002</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.2 Preparation of the surgery material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Forceps - DeBakey Pattern- 12.5 cm</td>
			<td>FST</td>
			<td>11617-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Arched tip Forceps- 10 cm</td>
			<td>FST</td>
			<td>11071-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bead sterilizer Steri 250</td>
			<td>Sigma</td>
			<td>Z378569</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass micropipette 1 mm diameter</td>
			<td>FHC</td>
			<td>10-10-L</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps - Titanium 1 mm Tips Slight Curve- 10 cm</td>
			<td>FST</td>
			<td>11651-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps - Titanium 1 mm Tips Straight- 10 cm</td>
			<td>FST</td>
			<td>11650-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight- 9 cm</td>
			<td>FST</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory tape</td>
			<td>Fisher Scientitic</td>
			<td>11730454</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>INJECT+MATIC</td>
			<td>No catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder - 12 cm</td>
			<td>FST</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>VWR</td>
			<td>631-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic-coated white paper</td>
			<td>Distrimed</td>
			<td>700103</td>
			<td></td>
		</tr>
		<tr>
			<td>Signagel electrode gel</td>
			<td>Free-Med</td>
			<td>15-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dish- 35 mm</td>
			<td>Dutscher</td>
			<td>056714</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer, glass dry bead, Steri 250</td>
			<td>Sigma</td>
			<td>Z378569</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.3 Preparation of the pregnant female mouse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol pad</td>
			<td>Alcomed</td>
			<td>1731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprecare</td>
			<td>Axience</td>
			<td>0.3 mg/ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Compress</td>
			<td>tRAFFIN</td>
			<td>70189</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Merial</td>
			<td>Imalgene 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular gel</td>
			<td>tvm lab</td>
			<td>Ocry-gel</td>
			<td></td>
		</tr>
		<tr>
			<td>RjOrl:SWISS mice</td>
			<td>Janvier Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vetadine, 10% solution</td>
			<td>Vetoquinol</td>
			<td>4337400113B</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming pad</td>
			<td>Harvard Apparatus</td>
			<td>72-0493</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>Rompun 2%</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable suture Size 4-0 45 cm Suture 1-Needle 19 mm Length 3/8 Circle Reverse</td>
			<td>Novosyn</td>
			<td>C0068220</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporateur Sonidel</td>
			<td>Sonidel</td>
			<td>NEPA 21</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile transfer pipets (individually wrapped)</td>
			<td>Dutscher</td>
			<td>043202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers with 3 mm platinium disk electrodes</td>
			<td>Sonidel</td>
			<td>CUY650P3</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4.1 Tissue harvesting and sectioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigenfix</td>
			<td>Microm Microtech</td>
			<td>U/P0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Dutscher</td>
			<td>100266</td>
			<td></td>
		</tr>
		<tr>
			<td>Dolethal</td>
			<td>Vetoquinol</td>
			<td>DOL202</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (10X), no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>11540486</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>EMS</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Dutscher</td>
			<td>100001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vectorlabs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5. Multichannel confocal imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X oil NA 0.85</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM880</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apochromat 20x/0.8 M27</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6. Astrocyte territorial volume segmentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMARIS 8.3 and later versions</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7. Astrocyte arborization tracing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D Visualization-Assisted Analysis software suite (Vaa3D)</td>
			<td>HHMI - Janelia Research Campus /Allen Institute for Brain Science</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in utero electroporation of multiaddressable genome-integrating color (magic) markers to individualize cortical mouse astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

Electroporation of MAGIC Markers in embryonic cortical progenitors allows for the labeling of astrocytes from early to late stages of cerebral cortex development (Figure 1). These astrocytes were found in all cortical layers at various postnatal stages (P4, P7, P21) as they dispersed widely into the entire cerebral cortex. They were assessed with tiled confocal images acquired with a 20x 0.8 NA (or higher NA) objective and assembled as Z-stack reconstructions (Figure 2</...

Watch this Scientific Journal Video about In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes at JoVE.com

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

In Utero electroporation of MAGIC Marker is a powerful and versatile technique for quickly individualizing and assessing the morphology of cortical astrocyte. The main advantage of this particular technique is to label multiple progenitor cells and the neuronal and viral progeny with a precise control. Demonstrating the procedure with me will be Karine Loulier, the principal investigator from Before beginning the procedure, sterilize all of the surgical tools at a high temperature in a glass bead sterilizer.And place a drop of electrode gel into a 35 millimeter dish. Insert a micropipette into a micro injection holder and break the tip of the micropipette. Aspirate the DNA solution and place a one microliter drop a fast green solution with a pipette into the lid of a three centimeter dish as reference.Then add a volume from the broken micropipette next to the reference drop and readjust the pressure or time parameter if necessary;so that the equivalent size drops will be produced by the micro injector. If the volume is smaller than the reference one, break the tip of the micropipette again and repeat. To prepare the pregnant mouse, after weighing and anesthetic injection, confirm a lack of response in the anesthetized female mouse and apply ointment to the animal's eyes.Gently shaved the abdomen of the mouse placed in the supine position on a warming pad. Wipe the exposed skin with one pad soaked with iodine and one pad soaked with alcohol. And place sterile compresses around the shaved, cleaned and sanitized surgical area of the animal.When the mouse has been prepped make a two centimeter vertical midline incision in the lower abdomen. And gently manipulate the embryonic bags to expose the uterine horns. Assess the location of the cervix and the number of embryonic bags on each side of the cervix.And orient the brain of the embryonic day 15.5 embryo to be electroporated so that the brahma, which is easily recognized at the junction of the three main blood vessels that run along the cerebral fissures, can be visualized. Imagining a virtual line between the brahma and the eye, introduce the micropipette between the virtual line and the longitudinal fissure. Then press the foot pedal to deliver one microliter of DNA solution into the lateral ventricle of the targeted hemisphere.A successfully injected ventricle will appear blue. After the DNA has been injected apply electrodes on both sides of the embryo with the anode covering the injected hemisphere. Press the foot pedal of the electroporator to deliver a series of four 50 millisecond 35 volt charges, separated by 950 millisecond intervals.Then hydrate the embryo with warm saline solution. When all of the targeted embryos have been electroporated return the uterine horns to the abdomen and fill the abdominal cavity with warm saline. Close the abdominal muscle with a continuous absorbable suture.And close the skin with approximately 10 individual stitches of a 4-0 absorbable suture. At the end of the surgical procedure, place the skin sutures regularly along the incision. Then place the animal in a clean cage with monitoring until full recovery.The fully recovered pregnant mouse should have a neat hair coat and an intact surgical incision. For multi-channel confocal imaging of the injected tissues, set the microscope for excitation of the appropriate lasers. And locate the brightest astrocytes in the electroporated area.Because the labeled cells closest to the surface may appear brighter than those deeper within the slice, adjust the acquisition settings on the surface cells to avoid saturating the images, using the high-low lookup table. An adequately balanced image should display only a few blue and almost no red pixels. Then use the motorized stage of the microscope to acquire tiled 1024-by-1024 pixels Z stacks with a 10%overlap between adjacent stacks to subsequently enable mosaic reconstructions of the electroporated area or the zone of interest.The labeled astrocytes can be assessed with tiled confocal images acquired with a 20x, 0.8na objective and assembled as Z stack reconstructions as demonstrated. The contour of individual astrocytes can be delineated on each individual optical section of the confocal image stacks for segmentation and reconstruction of the territorial domain occupied by each astrocyte. From the same Z image stacks, the branch morphology of singled out cortical astrocytes can be segmented using open access software that allows extraction of the skeleton of the main astrocyte processes.Proper hydration during the surgery and before the closing suture are placed is critical to boost the survivor of the embryos and the recovery of the mother. In Utero electroporation of MAGIC Marker toolbox can be used to label progenitor cells in distinct model at value stages of development to trace lineage at the single cell resolution.

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...