Name: intraventricular transplantation of engineered neuronal precursors for in vivo neuroarchitecture studies
Uploaded: 2019-05-11T12:30:00
Description: Watch this Scientific Journal Video about Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies at JoVE.com

We thank Mihn Duc Do for his contribution to early setting up of this procedure.

All methods and procedures described here have been approved by the SISSA Organismo preposto al Benessere Animale (SISSA IACUC).
1. Generation of engineered &#34;green&#34; progenitor pools
<ol>
	<li>Preparation of the &#34;green&#34; pool
	<ol>
		<li>Mate a wild-type CD1 female with a MtaptEGFP/+&#160;founder16. Euthanize the pregnant dam by cervical dislocation at 12.5 days post-coitum (day 0 is determined by vaginal plug inspect...

<ol>
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V., 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=A+CaMKII&#946;+2+signaling+pathway+at+the+centrosome+regulates+dendrite+patterning+in+the+brain.">A CaMKII&#946; 2 signaling pathway at the centrosome regulates dendrite patterning in the brain.</a> Nature Neuroscience. , (2011).</li><li>Zuccotti, A., Le Magueresse, C., Chen, M., Neitz, A., Monyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Fezf2+directs+the+differentiation+of+neural+stem+cells+in+the+subventricular+zone+toward+a+cortical+phenotype.">The transcription factor Fezf2 directs the differentiation of neural stem cells in the subventricular zone toward a cortical phenotype.</a> Proceedings of the National Academy of Sciences. , (2014).</li><li>Mikuni, T., Nishiyama, J., Sun, Y., Kamasawa, N., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput,+High-Resolution+Mapping+of+Protein+Localization+in+Mammalian+Brain+by+in+Vivo+Genome+Editing.">High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by in Vivo Genome Editing.</a> Cell. , (2016).</li><li>Spiga, S., Acquas, E., Puddu, M. C., Mulas, G., Lintas, A., Diana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Golgi-Cox+and+immunofluorescence+using+confocal+microscopy.">Simultaneous Golgi-Cox and immunofluorescence using confocal microscopy.</a> Brain Structure and Function. , (2011).</li><li>Chen, B., 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=The+Fezf2-Ctip2+genetic+pathway+regulates+the+fate+choice+of+subcortical+projection+neurons+in+the+developing+cerebral+cortex.">The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex.</a> Proceedings of the National Academy of Sciences. , (2008).</li><li>Feng, G., 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. , (2000).</li><li>Lu, X. H., Yang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically-directed+Sparse+Neuronal+Labeling+in+BAC+Transgenic+Mice+through+Mononucleotide+Repeat+Frameshift.">Genetically-directed Sparse Neuronal Labeling in BAC Transgenic Mice through Mononucleotide Repeat Frameshift.</a> Scientific Reports. , (2017).</li><li>Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+tools+for+the+Brainbow+toolbox.">Improved tools for the Brainbow toolbox.</a> Nature Methods. , (2013).</li><li>Garcia-Moreno, F., Vasistha, N. 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Specific aspects/steps of this procedure are critical and require special attention. First, (a) operators must be adequately pretrained to safely manipulate lentiviruses in a BSL-2 compliant lab environment. Second, (b) prior to mix &#34;test&#34; and &#34;control&#34; neural preparations, it is mandatory to carefully wash the two corresponding neurosphere suspensions as described, in order to prevent any delayed cross-infection of the two preparations due to unwanted lentiviral carry over. Third, (c) while transplanting...

Here we describe a simple method we developed to dissect in vivo gene control of neuronal cytoarchitecture. Based on in vitro engineering of neuronal precursors, their transplantation into neonatal brain and paired morphometric evaluation of &#34;test&#34; and &#34;control&#34; cells, it allows to unveil functional implications of test genes in fine control of neuronal morphology in a fast and affordable way. To investigate in vivo&#160;gene control of neuronal architecture, three key technical issues must be addressed: (1) achieving an adequately patterned gene-of-interest (GOI) expression and an accurate quantitative control of it; (2) obtaining a properly segmented visualization of distinct neuronal silhouettes; (3) eliciting statistical significance of results while employing a limited number of animals.
When available, mouse mutant lines harboring tetracycline (tet)-controlled transgenes may be the best tool to address the first issue1. Alternatively, somatic transgenesis may be employed. In such cases, the transgene is delivered via electroporation2 or viral transduction3. Next, it is retained as an episome (e.g., upon standard electroporation4), or it is integrated into the genome (randomly, via retroviral integrase5; or in a defined location, via CRISPR-promoted homologous recombination (SLENDR)6).
Second, neuronal silhouette visualization may be achieved via (a) sparse uniform labeling or (b) dense differential labeling. As for sparse labeling, advanced Golgi-like methodologies may be employed7, selected neuronal minisets can be filled by biocytin8, and salt-and-pepper labeling can be obtained thanks to a sparsely expressed transgene. Such a transgene may display variegated transcription (Thy-EGFP)9 or may be activated by stochastic recombination (MORF)10. As for (b), state of art strategies include Cre-mediated stochastic recombination within a multi-floxed fluoroprotein transgene array (Brainbow)11, as well as piggyBac-transposase-driven genomic integration of fluoroprotein genes, previously delivered via somatic transgenesis (CLoNE)12.
As for the third issue, the morphometric outcome is often affected by a large random variability, originating from inter-animal differences and cell-injection contingencies. Because of this, a large number of animals is usually employed to achieve the statistical power necessary to assess GOI morphometric activity.
The approaches previously described often rely on advanced technical skills and require conspicuous financial resources, which may limit their diffusion within the scientific community. To circumvent these issues, we conceived an easy and straightforward pipeline to dissect gene control of neuroarchitecture in vivo in a fast and affordable way. This is inspired by a similar co-transplantation design previously developed for fast in vivo evaluation of antiblastic transgene activity13.
Specifically, it is thought that the co-transplantation of in vitro engineered &#34;green&#34; neural precursors (&#34;test&#34; and &#34;control&#34; cells) into a &#34;black&#34; recipient neonatal brain can simultaneously fix the three key issues listed above. In fact, in vitro lentiviral engineering of precursors, in well-controlled conditions, allows maintenance of variability of neuronal transgene expression at a minimum, far below that usually associated with in vivo somatic manipulations (performed previously14,15 and in our unpublished results). The resulting accurate control of gene expression is comparable with that achieved by tet-controlled transgenic models. However, costs of this procedure are far below those originating from maintenance of a transgenic mouse line. Next, free-hand cell injection is easy and requires minimal training. Moreover, the amount of labeled precursors injected into each brain can be easily tuned to achieve a sufficient cumulative number of sparsely distributed precursors, while also keeping the total number of transplanted animals at a minimum. Last but not least, co-injection of differently fluoro-labeled, &#34;test&#34; and &#34;control&#34; precursors and subsequent pairwise statistical analysis of the results counteract the effects of inter-animal experimental variability, allowing the reaching of statistical significance of results, even upon the analysis of a limited number of individuals13.
It should be emphasized that, albeit fast and cheap, this method has two main limitations. First, it is designed to investigate cell-autonomous gene control of neuronal architecture, and it is not appropriate to address environmental control. Second, as transplanted neuronal precursors reach their final location by a heterochronic schedule, this method is preferable to model neuroarchitectonics control occurring past migration completion.

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			<td height="21" style="height:21px;width:487px;">0.3 mL syringe</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">320840</td>
			<td style="width:247px;">store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;m sterile filter</td>
			<td>Millex-HV</td>
			<td>SLHU033RS</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 multiwell plate</td>
			<td>Falcon</td>
			<td>353043</td>
			<td>store at RT</td>
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		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 multiwell plate</td>
			<td>Falcon</td>
			<td>351147</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EGFP antibody, chicken polyclonal, RRID:AB_371416</td>
			<td>Tebubio</td>
			<td>GTX13970</td>
			<td>store at -20 &#8451;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">anti-RFP antibody, rat monoclonal, RRID:AB_10795839</td>
			<td>Antibodies Online</td>
			<td>ABIN334653</td>
			<td>store at +4 &#8451;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">anti-Tubb3 antibody, mouse monoclonal, RRID:AB_2313773</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aspirator tube assemblies kit for calibrated microcapillary pipettes</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue light lamp</td>
			<td>Nightsea</td>
			<td>BLS2</td>
			<td>store at RT</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillaries</td>
			<td>Kwik-Fil</td>
			<td>TW150-4</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td>store at +4 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;">B&#252;rker chamber</td>
			<td>Sigma</td>
			<td>BR719520</td>
			<td>0.0025 mm2, 0.100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryo-inclusion medium (Killik)</td>
			<td>Bio-Optica</td>
			<td>05-9801</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>store at +4 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable embedding mold</td>
			<td>Bio-Optica</td>
			<td>07MP7070</td>
			<td>DispoMold07</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td>store at +4 &#8451;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dnase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>store at +4 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycicline</td>
			<td>Sigma</td>
			<td>D1822</td>
			<td>store at +4 &#8451;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps #3c</td>
			<td>Fine Science Tools</td>
			<td>11231-20</td>
			<td>store at RT</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGF</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>Gibco</td>
			<td>PHG0261</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td>store at RT</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fungizone (Amphotericin B)</td>
			<td>Gibco</td>
			<td>15290018</td>
			<td>store at -20 &#8451;</td>
		</tr>
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			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>store at RT</td>
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		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-chicken Alexa 488</td>
			<td>Invitrogen</td>
			<td>A11039</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rat Alexa 594</td>
			<td>Invitrogen</td>
			<td>A11007</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin Solution</td>
			<td>Stem Cell Technologies</td>
			<td>07980</td>
			<td>store at +4 &#8451;</td>
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			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>store at -20 &#8451;</td>
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			<td height="21" style="height:21px;">Optical fibers</td>
			<td>Leica</td>
			<td>CLS150X</td>
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			<td height="21" style="height:21px;">P1000 puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000 model</td>
			<td>Flaming/Brown Micropipette Puller</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen Strep</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #363 [LV_TREt_(IRES)PLAP]</td>
			<td>built in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #386 [LV_pTa1_mCherry]</td>
			<td>built in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #401 [LV_pTa1_rtTA(M2)]</td>
			<td>built in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #408 [LV_Ppgk1p_rtTA(M2)]</td>
			<td>built in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #484 [LV_lacZ]</td>
			<td>Addgene</td>
			<td>12108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #529 [LV_Pgk1p_mCherry]</td>
			<td>built in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid #730 [LV_pSyn_rtTA(M2)]</td>
			<td>built in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel</td>
			<td>Braun</td>
			<td>BB515</td>
			<td>store at RT</td>
		</tr>
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			<td height="21" style="height:21px;">Steromicroscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>store at -20 &#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td>store at +4 &#8451;</td>
		</tr>
	</tbody>
</table>

intraventricular transplantation of engineered neuronal precursors for in vivo neuroarchitecture studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

There are five primary datasets providing useful information about key aspects of the procedure, the first being (1) efficiency of neural precursors transduction and co-transduction by lentiviral vectors. (2) An example of key features of the promoters employed to drive the &#34;test gene&#34;. (3) An example of engineered cells ready for transplantation. (4) A cartoon including key procedural details of cell microinjection into the neonatal brain. (5) A synopsis of the whole morphometric...

Watch this Scientific Journal Video about Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies at JoVE.com

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

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