This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft).

1. Preparation of DNA and Glass Micropipettes for Injection
<ol>
	<li>Good quality glass micropipettes are essential to reduce initial high abortion rate due to loss of amniotic fluid. The procedure to pull glass micropipettes has been well documented13,18,25. Use 1.2 mm diameter capillaries pulled in a conventional Sutter P-97 device with the settings P=500; Heat=300; Pull=40; Velocity=50; Time=50. Fit the puller with 3 mm "trough" filaments (Sutter Instrument FT330B). The 2 mm size filaments have yielded f...

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1, E203-E207 (1999).</li><li>Miyasaka, N., Arimatsu, Y., Takiguchihayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foreign+gene+expression+in+an+organotypic+culture+of+cortical+anlage+after+in+vivo+electroporation.">Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.</a> Neuroreport. 10, 2319-2323 (1999).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev. Biol. 240, 237-246 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+tend+to+stop+migration+and+differentiate+along+the+cortical+internal+plexiform+zones+in+the+Reelin+signal-deficient+mice.">Neurons tend to stop migration and differentiate along the cortical internal plexiform zones in the Reelin signal-deficient mice.</a> J. Neurosci. Res. 69, 723-730 (2002).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emx2+patterns+the+neocortex+by+regulating+FGF+positional+signaling.">Emx2 patterns the neocortex by regulating FGF positional signaling.</a> Nat. Neurosci. 6, 825-831 (2003).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+8+regulates+neocortical+guidance+of+area-specific+thalamic+innervation.">Fibroblast growth factor 8 regulates neocortical guidance of area-specific thalamic innervation.</a> J. Neurosci. 25, 6550-6560 (2005).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Dixit, R., 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=Efficient+Gene+Delivery+into+Multiple+CNS+Territories+Using+In+Utero+Electroporation.">Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation.</a> J. Vis. Exp. (52), e2957 (2011).</li><li>Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> J. Vis. Exp. (44), e2103 (2010).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Intraventricular+Injection+and+Electroporation+of+E15+Mouse+Embryos.">In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos.</a> J. Vis. Exp. (6), e239 (2007).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Intraventricular+Injection+and+Electroporation+of+E16+Rat+Embryos.">In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos.</a> J. Vis. Exp. (6), e236 (2007).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> J. Neurosci. Methods. 143, 151-158 (2005).</li><li>Haddad-Tovolli, R., Heide, M., Zhou, X., Blaess, S., Alvarez-Bolado, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+thalamic+differentiation:+gli-dependent+pattern+and+gli-independent+prepattern.">Mouse thalamic differentiation: gli-dependent pattern and gli-independent prepattern.</a> Front Neurosci. 6, 27 (2012).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+Utero+Electroporation:+Controlled+Spatiotemporal+Gene+Transfection.">Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection.</a> J. Vis. Exp. (54), e3024 (2011).</li><li>Vue, T. Y., 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=Sonic+hedgehog+signaling+controls+thalamic+progenitor+identity+and+nuclei+specification+in+mice.">Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice.</a> J. Neurosci. 29, 4484-4497 (2009).</li><li>Tanaka, S., 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=Transcriptional+regulation+of+the+hypocretin/orexin+gene+by+NR6A1.">Transcriptional regulation of the hypocretin/orexin gene by NR6A1.</a> Biochem. Biophys. Res. Commun. 403, 178-183 (2010).</li><li>Tsuchiya, R., Takahashi, K., Liu, F. C., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+axonal+projections+from+mammillary+bodies+in+Pax6+mutant+mice:+possible+roles+of+Netrin-1+and+Slit+2+in+mammillary+projections.">Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections.</a> J. Neurosci. Res. 87, 1620-1633 (2009).</li><li>Canteras, N. S., Watson, C., Paxinos, G., Puelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Nervous System. , 539-562 (2011).</li><li>Caqueret, A., Yang, C., Duplan, S., Boucher, F., Michaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+for+trouble:+a+search+for+developmental+defects+of+the+hypothalamus.">Looking for trouble: a search for developmental defects of the hypothalamus.</a> Horm. Res. 64, 222-230 (2005).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+Electroporation+for+Gene+Expression+in+Mouse+Retinal+Ganglion+Cells.">In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells.</a> J. Vis. Exp. (31), e1333 (2009).</li><li>Nijagal, A., Le, T., Wegorzewska, M., Mackenzie, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+of+in+Utero+Transplantation.">A Mouse Model of in Utero Transplantation.</a> J. Vis. Exp. (47), e2303 (2011).</li><li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+of+the+Rat+Hypothalamus.">The Development of the Rat Hypothalamus.</a> Adv. Anat. Embryol. Cell Biol. 100, 1-178 (1986).</li><li>Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105, 7-17 (2001).</li><li>Clancy, 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=Web-based+method+for+translating+neurodevelopment+from+laboratory+species+to+humans.">Web-based method for translating neurodevelopment from laboratory species to humans.</a> Neuroinformatics. 5, 79-94 (2007).</li><li>Ishii, Y., Bouret, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+birthdate+of+hypothalamic+leptin-activated+neurons+in+mice.">Embryonic birthdate of hypothalamic leptin-activated neurons in mice.</a> Endocrinology. 153, 3657-3667 (2012).</li><li>Karim, M. A., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+supraoptic+and+paraventricular+neurosecretory+cells+of+the+mouse+hypothalamus.">Histogenesis of the supraoptic and paraventricular neurosecretory cells of the mouse hypothalamus.</a> J. Anat. 130, 341-347 (1980).</li><li>Shimada, M., Nakamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+of+neuron+origin+in+mouse+hypothalamic+nuclei.">Time of neuron origin in mouse hypothalamic nuclei.</a> Exp. Neurol. 41, 163-173 (1973).</li><li>Alvarez-Bolado, G., Paul, F. A., Blaess, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+lineage+in+the+mouse+hypothalamus:+from+progenitor+domains+to+hypothalamic+regions.">Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions.</a> Neural. Dev. 7, 4 (2012).</li><li>Vann, S. D., Aggleton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammillary+bodies:+two+memory+systems+in+one.">The mammillary bodies: two memory systems in one.</a> Nat. Rev. Neurosci. 5, 35-44 (2004).</li><li>Gratsch, T. E., De Boer, L. S., O'Shea, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+inhibition+of+BMP-4+gene+expression+in+postimplantation+mouse+embryos.">RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos.</a> Genesis. 37, 12-17 (2003).</li><li>Wu, N., Yu, A. B., Zhu, H. B., Lin, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+silencing+of+Sry+gene+with+RNA+interference+in+developing+mouse+embryos+resulted+in+feminization+of+XY+gonad.">Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.</a> J. Biomed. Biotechnol. 2012, 343891 (2012).</li><li>Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+mechanism+of+gene+transfer+into+early+to+mid-gestational+mouse+fetuses+by+tail+vein+injection.">Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.</a> Gene Ther. 9, 1529-1541 (2002).</li><li>Foust, K. D., 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=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Xin, 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=The+brain+targeting+mechanism+of+Angiopep-conjugated+poly(ethylene+glycol)-co-poly(epsilon-caprolactone)+nanoparticles.">The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles.</a> Biomaterials. 33, 1673-1681 (2012).</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, 193-199 (1991).</li><li>Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+stable+expression+of+foreign+genes+in+mammalian+cells+by+use+of+the+untranslated+leader+sequence+from+EMC+virus.">Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.</a> Nucleic Acids Res. 19, 4485-4490 (1991).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Londrigan, S. 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=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

The authors declare that they have no competing financial interests.

About the anesthesia: Since in utero electroporation into the hypothalamus can be technically arduous and require longer narcosis times, we prefer to induce and maintain anesthesia through administration of a mixture of oxygen and isoflurane. In our experience, animals can remain suitably anaesthetized in this way for periods of up to one hour at least, the recovery of the mother is very fast, and embryo survival improved. Other approaches to anesthesia are also available. The most simple procedure consis...

Genetic manipulation of the embryonic mouse brain is a preferred approach to learn about developmental regulation. The generation of mutant mouse lines however is slow and expensive. One powerful method to introduce specific genetic changes in developing neurons of the mammalian brain is in utero electroporation. Essentially, the technique consists of transfecting DNA into the embryonic brain neuroepithelium by means of electric pulses, then allowing the embryo to survive for a certain period of time, collect the brain and examine them for possible novel, informative phenotypes. In this way, the experimenter can test hypotheses almost immediately without the long waiting periods necessary for the production of mouse mutants.	
	Transfection of DNA into developing embryos started with in ovo electroporation on chick embryos1. The essential proof-of-concept for the mouse was performed in culture2. This was soon followed by the first descriptions of the technique on the mouse in utero3,4.	
	The main problem is to transfect the brain of embryos developing in utero without killing them or the mother. Learning to perform the necessary surgery (laparotomy, injection, electroporation) requires a long training period. Once the surgery has been mastered to the point where the embryo survival ratio is acceptable, the next key question is: which brain structures are accessible? Not surprisingly, the first published papers showing results obtained with in utero electroporation focused on cortical development5-9. This is still true for most of the publications using this technique, since the region of the developing mouse brain most accessible to surgical procedures is the cortex (Figure 1). The procedure for in utero electroporation into the cortex has been described in print10 and in video11-14. A modification of the technique can be used to target a ventral part of the telencephalon, the basal ganglia15.	
	Beyond the telencephalon, the diencephalon (classically divided into thalamus and hypothalamus) is a region of the forebrain more difficult to reach. A small number of papers reports targeting of its dorsal and most accessible part, the thalamus16-19.	
	The hypothalamus is the most ventral part of the forebrain, therefore the one localized most deeply from the dorsal surface (cortex) (Figure 1). This region remains a difficult challenge for researchers committed to genetically manipulate the mouse brain in utero. To our knowledge, only very few articles report on results of in utero transfection into the mouse hypothalamus 20,21. However, the functional importance of the hypothalamus cannot be overstated, since it regulates behaviors like eating and drinking, mating, breeding and parenting22. Moreover, alterations in hypothalamic development contribute to originate later in life conditions like obesity, hypertension, diabetes and precocious puberty23. Being able to alter genetically the hypothalamus during development would provide a very powerful tool to understand it.	
	The basic surgical protocol for the laparotomy of pregnant mice that we use here is similar to that used in other protocols11,13,14,24. We will describe them here briefly for completeness. Key to our procedure, on the other hand, are the type of anesthesia, the place of injection, the type of electrodes and the insertion and position of the positive electrode with respect to the embryo's head. We prefer to induce and maintain anesthesia through gas inhalation over simple intraperitoneal anesthesia, since the former allows for the somewhat longer periods of narcosis required for a difficult surgery. Isoflurane inhalation results in faster recovery from anesthesia, since usually the mother demonstrates normal behavior already minutes after the surgery. The easiest point of injection of the DNA solution with the glass micropipette is the lateral ventricle, which however is completely unsuitable for hypothalamus electroporation. Injection directly in the third ventricle is indeed crucial to target deep diencephalic structures. It is possible to transfect the hypothalamus from E12.0 or E12.5 with standard, off-the-shelf electrodes. We have found some of the electrodes manufactured by Nepa Gene (Chiba, Japan) particularly suited to this purpose.	
	With our procedure we obtain transfection of the entire hypothalamic neuroepithelium or partial, regional transfection depending on electrode orientation. Here we demonstrate the technique by transfecting the mammillary body, arguably the deepest and most recessed of all hypothalamic nuclei. Additionally, we show detailed histological analysis of the transfected cells down to the cellular level of resolution.	
	A comparison of in utero electroporation with other approaches to transfecting the mouse developing brain in utero can be found in the Discussion section.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

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 mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Most hypothalamus neurons are born between E11.5 to E15.2, according to birth-dating analysis in the rat26 translated into the somewhat shorter mouse development27,28. The peak of hypothalamic neurogenesis is reached at E12.529-31. Accordingly, at the transfection age chosen for the present study (E12.5), a large proportion of hypothalamic neurons can be labeled at any given rostro-caudal level.
Analysis at E18.5 on thick vibratome-type sections (Figur...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

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 ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

15.7K Görüntüleme.  University of Heidelberg. Hipotalamusun fonksiyonel ve tıbbi önemine rağmen, Utero genetik manipülasyon nadiren çalışılmıştır. Biz için ayrıntılı bir prosedür göstermek Utero elektroporasyon.