We thank Bettina Semsch and Jia Sun (Infinigene) for expert care of mice; Florian Salomons and G&#246;ran M&#229;nsson from Biomedicum Imaging Core (BIC) for assistance with image acquisition and consultation. Funding: We thank the following funders for their support of this project: The Swedish Research Council, Karolinska Institutet (KI Foundations, Career Development Grant, Ph.D. student KID funding, and SFO StratNeuro funding, the Center of Innovative Medicine), The Ollie and Elof Ericssons Foundation, the Tornspiran Foundation, the Jeansssons Foundation, Sven and Ebba-Christina Hagbergs Prize and research Grant, Knut and Alice Wallenberg Project Grant, Fredrik and Ingrid Thurings Foundation, Lars Hiertas Minne, The Childhood Cancer Foundation (Barncancerfonden), The &#197;hlen Foundation, &#197;ke Wibergs Foundation, Tore Nilssons Foundation, and the Swedish Foundations Starting Grant to ERA. Figure 4D,E were created with BioRender.com.

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</ol>

The authors declare no competing interests.

There are several steps in this protocol that influence embryonic survival, the quality of the injections, and the readout. The gestational age of embryos is defined as E0.5 at noon the day of the vaginal plug after overnight mating. Performing the ultrasound check for pregnancy at E6.5 in the late afternoon/evening ensures that the embryos are developed enough to be identified by ultrasound. The check (1) allows for prescreening of how many plug-positive mice are actually pregnant, (2) ensures no virus is thawed unnecessarily and wasted in the event of plug-positive mice not being pregnant, and (3) reduces unnecessary interventions on mice (avoids surgery on non-pregnant females).
At E7.5, embryos are sensitive to external forces and should be handled with care. For example, pulling on the uterine horns or squeezing the deciduas can lead to embryo resorption. The uterine tissue should always be kept moist when outside the female abdomen to prevent the tissue from drying. The majority of the deciduas should remain inside the female abdomen, with only 3-4 exposed for injections. Needle sharpness is another crucial determinant for successful injections. Blunt or broken needle tips result in repeated poking of deciduas or compression against the modeling clay before entering the AC, which can increase the resorption rate. Therefore, well-ground and sharp needles should always be stored safely and replaced after a maximum of 2 females.
This protocol describes how to target the neural plate with one single injection of lentivirus. Furthermore, it shows how transduction efficacy can be adapted from single-cell clones to the entire brain. However, other non-neural tissues, including the skin and oral epithelium, are targeted as well. In addition, all cell types (progenitors and differentiated cells) are transduced, making this approach efficient but nonspecific. The use of MiniPromoters in the viral construct leads to the specific expression of the transgene in neurons or astrocytes15. This has the advantage of avoiding the use of dedicated transgenic Cre animals and therefore reduces the amount of labor (strain maintenance and genotyping) and costs.
The limitations of NEPTUNE include its technical difficulty, challenges in obtaining pregnant females at a predictable and consistent rate, and the costs of acquiring specialized instrumentation. Furthermore, nonselective targeting of cells by lentivirus can be seen as both a benefit and a limitation of the technique. Injection of larger volumes into the AC results in exencephaly13, although brain malformations and exencephaly are avoided with the volumes described here15. A negative impact on brain development is thus a risk with in utero nano-injections that must be carefully avoided by injecting correct volumes adapted to the embryonic stage and AC size.
Future adaptations of the technique may focus on viral tropism. Adeno-associated viruses (AAVs) have different serotypes, which have been shown to robustly target different cell types in the CNS17,26. However, AAVs do not integrate into the host cell genome and therefore may be lost in cells with a high division rate. Although there are several ways to increase the specificity of NEPTUNE, transgenic animals are still the gold standard when it comes to gene manipulation in vivo. Cas9 mice and sgRNA-encoding lentivirus have been used for genetic screening in the embryonic epidermis27 and may also be adapted to the developing CNS.
Injections into the AC at E7.5 efficiently target cells of the neuroectoderm before initiation of neurulation and targets the developing brain more efficiently than in utero electroporation. This allows the study of genetic cues important for brain development from an earlier time point. In contrast to classical genetic mouse models, NEPTUNE offers a flexible approach to perform functional gene analysis. Phenotypes following overexpression or gene deletion can be studied within days to weeks compared to months or years. Injections of multiple viral constructs allow for the manipulation of several genes within one embryo and avoid the generation of double or triple knockout animals. Therefore, NEPTUNE not only saves time but also can reduce the number of animals used in research.

The brain and spinal cord are among the first organs to initiate formation during embryogenesis1,2. Although the genes associated with neurodevelopmental disorders are being identified, the functional interrogation of genetic variants has lagged behind3,4. As the generation of conditional knockout mice can take months or years, an alternative technique to rapidly investigate gene function in the developing brain is of interest. In mouse embryos, neurulation - the morphogenetic process by which the neural plate transforms into the neural tube to give rise to the central nervous system (CNS) - occurs between days 8 and 10 post conception5. Prior to the onset of neurulation, the neural plate, as part of the ectoderm, consists of a single layer of columnar cells that will proliferate and differentiate into the numerous neuronal and glial cell types within the CNS6,7. Therefore, to experimentally induce long-lasting alterations to gene expression in the CNS, targeting the neural plate offers obvious advantages, including the accessibility of all progenitor cells.
In neuroscience, in ovo electroporation8,9 and viral transduction of mouse embryos have been used to manipulate embryonic CNS gene expression. The developing chick embryo has been a model of choice for studying gene function during spinal cord development due to the accessibility of the chick embryo in the egg and the resultant ease of manipulating gene expression. In particular, in ovo plasmid electroporation generates experimental and control conditions in each chick spinal cord. Electroporation causes cell membrane permeabilization and directs negatively charged DNA away from the (negative) cathode towards the (positive) anode by applying an electric pulse via two electrodes to the embryo. In mice, in utero electroporation has generally been limited to embryonic stages at which neurulation has completed, and the brain or spinal cord already consists of several cell layers, resulting in low electroporation efficiency10. Plasmid electroporation results in transient gene expression and generally targets few cells.
Ultrasound-guided in utero microinjection has been used to manipulate different embryonic structures such as the skin and brain11,12,13,14. However, injections targeting the developing murine CNS have shown low efficacy or have negatively impacted embryonic survival12,13,14. Therefore, an improved protocol was developed for the delivery of high-titer lentivirus into the amniotic cavity (AC) at E7.5, which was dubbed NEPTUNE for neural plate targeting with in utero&#160;nano-injection15. Injections resulted in a long-lasting targeting efficacy of &#62;95% of the entire brain at E13.5. Furthermore, a staging step was introduced during ultrasound verification of pregnancy to sort females and pregnancies by developmental stage to minimize unnecessary procedures on research animals and maximize injection success. Injection efficiency and survival are tightly linked to the increase in AC size. Therefore, this paper describes how to measure AC size prior to injection to deliver a suitable volume to the AC that will not cause resorption of the embryo. NEPTUNE is a robust alternative to current in utero approaches and can be adapted for several uses, including, but not limited to, gain and loss of function studies, lineage tracing, or screening15,16.

<table><tbody><tr><td>1 mL Syringe</td><td>BD Bioscience</td><td>309628</td><td></td></tr><tr><td>27 G Needle</td><td>BD Bioscience</td><td>300635</td><td></td></tr><tr><td>3.5 inches capillaries</td><td>Drummond Scientific</td><td>3000203G/X</td><td>Were used to pull in house needles</td></tr><tr><td>70 MHz MS Series transducer</td><td>Visual Sonics</td><td>MS700</td><td></td></tr><tr><td>Aquasonic clear ultrasound gel</td><td>Parker Laboratories</td><td>Mar-50</td><td></td></tr><tr><td>Autoclip Applier 9 mm</td><td>Angthos</td><td>12020-09</td><td></td></tr><tr><td>CD1 mice</td><td>Charles River, Germany</td><td>Crl:CD1(ICR)</td><td>Females: from age of 8 weeks old&lt;br/&gt; Males: from the age of 12 weeks old</td></tr><tr><td>Cotton Swab</td><td>OneMed Sverige AB</td><td>120788</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190094</td><td></td></tr><tr><td>Dressing forceps delicate straight 13 cm</td><td>Agnthos</td><td>08-032-130</td><td></td></tr><tr><td>EG-400 Narishige Micropipette Grinder</td><td>Narishige</td><td>NA</td><td></td></tr><tr><td>EZ clips 9 mm</td><td>Angthos</td><td>59027</td><td>clips</td></tr><tr><td>Iris Scissors, Super Cut, straight, 9 cm</td><td>Agnthos</td><td>307-336-090</td><td></td></tr><tr><td>Isofluorane</td><td>Baxter Medical AB</td><td>EAN: 50085412586613</td><td>Purchased from Swedish Pharmacy</td></tr><tr><td>Kimwipes</td><td>Kimberly Clarke</td><td>7557</td><td></td></tr><tr><td>Membrane Tape</td><td>Visual Sonics</td><td>SA-11053</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Modeling Clay</td><td>Sense AB</td><td>10209</td><td></td></tr><tr><td>Mouse Handling Table</td><td>Visual Sonics</td><td>50249</td><td></td></tr><tr><td>Nanoject II Auto Injector Kit</td><td>Drummond</td><td>3-000-205A</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>HS234526C</td><td></td></tr><tr><td>Petri dish with central opening (low wall)</td><td>Visual Sonics</td><td>SA-11620</td><td></td></tr><tr><td>Petri dish, (&amp;Oslash;xH): 92 x 16 mm</td><td>Sarstedt</td><td>82.1472.001</td><td></td></tr><tr><td>Rely+On Virkon</td><td>DuPont</td><td>130000132037</td><td>disinfectant</td></tr><tr><td>Silicone membrane</td><td>Visual Sonics</td><td>SA-11054</td><td></td></tr><tr><td>Steri 250, hot bead sterilizer</td><td>Angthos</td><td>31100</td><td></td></tr><tr><td>Surgical Tape (1.25 cm x 9.14 m)</td><td>Medicarrier</td><td>67034</td><td></td></tr><tr><td>Vevo Compact Dual (Med. Air &amp;amp; O&lt;sub&gt;2&lt;/sub&gt;) Anesthesia System</td><td>Visual Sonics</td><td>VS-12055</td><td></td></tr><tr><td>Vevo Imaging Station 2</td><td>Visual Sonics</td><td>VS-11983</td><td></td></tr><tr><td>Vevo2100</td><td>Visual Sonics</td><td>VS-20047</td><td></td></tr><tr><td>Vicryl 6-0; C-3 needle, 45 cm purple filament</td><td>Agnthos</td><td>J384H</td><td></td></tr></tbody></table>

murine neural plate targeting by in utero nano-injection (neptune) at embryonic day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Embryos injected at E7.5 with hPGK-H2B-GFP lentivirus11,12 were collected at E13.5 and examined under the fluorescent dissection microscope (Figure 5A). Successful transduction of the neural plate results in embryos with strong expression of a fluorescent reporter in the brain mainly and in other ectoderm-derived tissues, e.g., the skin (Figure 5A,B). Injection of an excessively high volume (greater than the volumes recommended here, e.g., &#8805;500 nL) increases the pressure in the AC and can result in either complete resorption (data not shown) or neural tube defects such as exencephaly (Figure 5A). Successful injections at E7.5 result in uniform transduction from the forebrain to the hindbrain (Figure 5C-J).
Lentiviral titers around 2 &#215; 1010 infectious units (IFU) achieve over 95% targeting, while titers of ~1 &#215; 109 IFU achieve 15% targeting efficiency15. In addition, structures that have previously been difficult to target using electroporation, such as the choroid plexus17,18, are also targeted (Figure 5 E,F). Transduction efficacy can be modified by adjusting the viral titer delivered into the AC. Injections of low titer result in the transduction of single-cell clones (Figure 5C, Figure 5E, and Figure 5G,H) while usage of high-titer virus transduces nearly 100% of the entire brain (Figure 5D, Figure 5F, Figure 5H, and Figure 5J). Therefore, NEPTUNE can be used for either clonal transduction, lineage tracing, and genetic screening approaches or studying the global effects of gene overexpression or downregulation in the entire brain.
Mammalian eye development is the result of well-organized communication between three derivatives of the embryonic ectoderm: the neural retina (NR) and retinal pigment epithelium (RPE) are derived from the neuroepithelium of the ventral forebrain, while the surface ectoderm gives rise to the future lens and corneal epithelium. However, the central stroma and the posterior endothelium, the other two layers of the cornea, are derived from neural crest cells of the periocular mesenchyme19,20. Coronal sections through E13.5 embryos, injected at E7.5 with high-titer lentivirus showed, similar to the brain, high and uniform transduction of the neural tissue of the eye, as well as the lens, cornea, and mesenchyme (Figure 5K). As neurulation progresses, injections at E8.5 and E9.5 result in the continued targeting of the lens and corneal epithelium (Figure 5L and Figure 5N), while transduction of the neuroectoderm-derived tissues of the eye is less efficient at E8.5 (Figure 5L,M) or not targeted at E9.5 (Figure 5N).
While most viral particles infect the exposed tissues upon injection, some particles transduce non-ectodermal derived tissues that develop later (Figure 6A). Salivary glands and ducts develop around E11.5 from the oral epithelium21 and are targeted with NEPTUNE (Figure 6B). Following injections at E9.5, the lingual epithelium of the tongue is well transduced; however, the underlying mesenchyme is negative (Figure 6C; brackets denote transduced cells; asterisks denote autofluorescence signal not transduced cells). In addition, there are positive clusters within the lingual epithelium, separated by negative sections (Figure 6C, white arrowheads), suggesting transduction of the papilla surface. Neural crest cells have been described in the underlying tongue mesenchyme and within the lingual epithelium, where they are involved in the development of taste papillae and taste buds22. Indeed, injections at E7.5 result in widespread transduction of the tongue mesenchyme at E13.5 (Figure 6D), suggesting that early injections target neural crest cells, contributing to mesenchyme in the tongue.
In vertebrates, the dorsal root ganglia (DRGs) are a central component of the peripheral nervous system (PNS), as all somatosensory input from the body&#39;s periphery (temperature, pain, pressure) is transmitted to the brain via activation of the DRG neurons23. Both neurons and glial cells of the DRG are derived from trunk neural crest cells24. Lentiviral injection, in which the ubiquitous hPGK promoter controls expression of the fluorescent reporter, leads to widespread targeting of the central and peripheral nervous system (Figure 7A), transducing both neurons and progenitors in the spinal cord (Figure 7B), as well as the DRG (Figure 7C). Using a MiniPromoter for doublecortin25 makes it possible to limit GFP expression to neurons only (15 and Figure 7D,E).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63148/63148fig5v3.jpg" /> 
Figure 5: High efficiency or clonal transduction with NEPTUNE. (A) E13.5 embryos under a dissection microscope illuminated with standard lighting (left panel) and same embryos illuminated for GFP (right panel). Uninjected embryo on the far left, successfully injected embryo on the far right, yielding positive signal in the brain (rightmost embryo). Exencephalic embryo due to excess volume injected (middle embryo). (B) Skin and brain targeting with E7.5 injection. Scale bar = 50 &#181;m. (C, D) E13.5 confocal images of forebrain with low clonal transduction (C) or high-efficiency transduction (D). (C, E, G, I) Clonal transduction of different regions in brain. (D, F, H, J) High-efficiency transduction of different regions in brain. Scale bars = 200 &#181;m. Different transduction efficacies are representative of other areas of the CNS. (E, F) Choroid Plexus targeting, clonally (E) or with high efficiency (F). Scale bars = 200 &#181;m. (G, H) Clonal targeting (G) and high-efficiency (H) targeting of hindbrain, magnified in (I, J). Scale bars = 200 &#181;m. (K) GFP reporter expression in eye at E13.5 after in utero injection at E7.5 (L, M) GFP reporter expression in eye at E15.5 after in utero injection at E8.5 (L), boxed region magnified in (M), or at E9.5 (N). Autofluorescent blood vessels are marked with white stars. Scale bars = 100 &#181;m. Abbreviations: NEPTUNE = neural plate targeting with in utero&#160;nano-injection; C = Cornea; LE = Lens Epithelium; LF = Lens fibers; M = Mesenchyme; NR = Neural Retina; ON = Optic Nerve; RPE = Retinal Pigment Epithelium; GFP = green fluorescent protein; CNS = central nervous system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63148/63148fig5v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63148/63148fig06.jpg" /> 
Figure 6: In utero transduction of non-neural tissues. (A) Confocal image of E15.5 oral cavity. Embryo was injected with fluorescent reporter lentivirus at E9.5. Scale bar = 200 &#181;m. (B, C) (B) Magnification of inset panel; salivary duct epithelium transduced with virus. (C) Magnification of inset panel; dorsal lingual epithelium transduced with GFP reporter virus (white bracket). The underlying mesenchyme derived from neural crest cells is negative. White arrowheads indicate papillae with negative neural crest cells surrounded by virus-transduced epithelial cells (autofluorescent blood cells/vessels are marked with white stars). Scale bars = 50 &#181;m. (D) Schematic and confocal image of E13.5 tongue mesenchyme after injections with fluorescent reporter virus at E7.5. Scale bar = 50 &#181;m. Abbreviations: D = Dorsal; M = Mesenchyme; N = Nasopharynx; SLD = Sublingual Duct; SMD = Submandibular Duct; T = Tongue; V = Ventral; GFP = green fluorescent protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63148/63148fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63148/63148fig07.jpg" /> 
Figure 7: Transduction of neural crest-derived dorsal root ganglion cells. (A) NEPTUNE targeting at E7.5 allows targeting of both CNS and PNS. (B) Confocal image of E13.5 spinal cord and DRGs injected with hPGK-H2B-GFP reporter lentivirus at E7.5 shows transduction of both SOX2+ neural progenitors and NeuN+ neurons. Scale bar = 100 &#181;m. (C) Boxed region of B showing the DRG with GFP expression in SOX2+ (white arrows) and NeuN+ (white arrowheads) cell populations. Scale bar = 20 &#181;m. (D) Confocal image of E13.5 spinal cord and DRG injected with DCX-H2B-GFP lentivirus at E7.5, targeting only DCX+ cells. Scale bar = 100 &#181;m. (E) Boxed region of D showing DRG with GFP expression restricted to DCX+ neurons (white arrowheads). DCX- cells are negative for GFP (white arrows). Scale bar = 20 &#181;m. Abbreviations: NEPTUNE = neural plate targeting with in utero&#160;nano-injection; CNS = central nervous system; PNS = peripheral nervous system; DRGs = dorsal root ganglia; GFP = green fluorescent protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63148/63148fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5 at JoVE.com

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

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Universidad Científica del Sur

Research

JoVE Journal

Developmental Biology

3.5K Views.  Karolinska Institutet. In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development. 

Video: Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Neuroscience

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 neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

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

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biology

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

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 pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

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

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

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

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Summary

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Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations