CD1 wild-type mice were housed according to European regulations, with a standard day and night cycle with food and water ad libitum. CD1 females were mated with CD1 males overnight, and vaginal plugs were checked in the morning (E0.3). Only pregnant females were used for the injection. Ethical approval for all experiments described here was granted by the Swedish Board of Agriculture (Jordbruksverket).
1. Preparation of glass needles: needle pulling and grinding
NOTE: Although preground needles can be bought, pulling needles in-house allows for easy adjustments of needle length, bore, and bevel angle.
Figure 1: Needle preparation for E7.5 amniotic cavity injections. (A) Representative examples of a pulled but uncut glass capillary needle (left), a capillary needle cut at the optimal length for E7.5 injections (middle), and a capillary cut too short (right). (B) The grinder with uniform coverage of water, which is ready for needle grinding. (C–F) Representative examples of different needle tips. (C) Cut but unground needle tip mounted in grinder; (D) ideal grinding position with the needle tip just touching the grinder; (E) needle lowered too far and bending during the grinding process; (F) an ideal ground needle tip for E7.5 AC injections. (G) A ground needle tip showing the bore with an inside diameter of ~15 µm and an outside diameter of ~35 µm, which is suitable for E7.5 AC injection. The needle bore is shown as dashed lines. Outer diameter denoted with red arrowheads; inner diameter denoted with blue arrowheads. (H) Needle storage: Petri dish filled with pulled and ground needles. Two rows of modeling clay serve as holders. NOTE: For the ocular micrometer in C, F, G, 1 cm is divided into 100 pitches; the objective is 3x; therefore, 1 pitch= 10,000 µm/(100 × 3) ≈ 33.4 µm. Abbreviations: AC = amniotic cavity; ID = internal diameter; OD = outer diameter. Please click here to view a larger version of this figure.
2. Day before injection: prepare bench for ultrasound-verification of pregnancy
NOTE: All work should be carried out in a ventilated Biosafety Level 2 (BSL 2) bench when working with lentivirus. Ultrasound-verification of pregnancy can be performed on a ventilated bench.
3. Ultrasound check to confirm pregnancy
NOTE: This step can be performed the day before E7.5 Injections, at E6.5. See the discussion for details about the check for gestational age.
4. Ultrasound check for embryo staging
NOTE: This step is performed before the surgery and serves to stratify the pregnant females according to their AC sizes. This step is crucial at E7.5 when the aim is to target the developing CNS. At this early stage of development, a difference of a few hours in development significantly influences the size of the AC and the progression of neurulation.
Figure 2: Inspection and staging of amniotic cavities during ultrasound-check. (A) Overview of the rail system with attached ultrasound probe, nanoinjector, and heating table. The ultrasound probe can be moved in x, y, and z-planes to achieve optimal alignment with the female abdomen or the ACs. (B) Heating table can be moved via two wheels in x- and/or y-planes to allow precise scanning and assessment of the ACs, while the ultrasound probe can remain static. (C) Representative ultrasound images of E6.5 deciduas inside the female abdomen during ultrasound check to confirm pregnancy (white dotted outlines). No cavities have formed at this point; however, sometimes, the ectoplacental cone (white asterisks) is visible. Deciduas can be recognized by their spherical shape and distinguished from the intestine, which appears as one continuous tube. (D) Representative image sequence of the small intestine (whited dotted outlines), which is continuous in scanning through the lower abdomen. (E–G) Representative ultrasound images during cavity staging prior to E7.5 injections. The amniotic and exocelomic cavities have formed and are separated by the amnion. The ectoplacental cone serves as the main blood supply and appears as a bright spot in the ultrasound. The AC is most distal from the ectoplacental cone. (E) Ideal-sized ACs appear larger than the exocelomic cavity, while medium-sized cavities appear smaller (F). If no cavities are visible (G), this means either that the embryo is resorbed or has not reached E7.5 yet. Scale bars = 1 mm. Abbreviations: A = Amnion; AC = Amniotic Cavity; ExC = Exocelomic Cavity; EC = Ectoplacental Cone. Please click here to view a larger version of this figure.
5. Day of injection: prepare the BSL2 bench for surgery
6. Needle loading
Figure 3: Attaching the needle to the nanoinjector and solution loading. (A) Starting position before the needle is mounted onto the nanoinjector: metal plunger completely retracted and collet attached. (B) Under the collet, all three components for holding and securing the needle are shown in correct order (from left to right): sealing O-ring (thin and black), spacer (white), O-ring (black) with big hole (that the needle must pass through). To ensure an airtight connection, the glass needle is slid over the metal plunger (C) and pushed through the opening of the front O-ring until it reaches the spacer (D). (E–H) Loading of the solution into the needle. (E) One drop of solution is placed on a piece of parafilm on a plate lid. (F) Create an air bubble by pressing Fill before loading the solution and immerse the needle tip in the solution. (G) Solution is loading into the needle. (H) Solution is loaded into the needle. NOTE: The collet has been removed in (B–D) for visualization but should remain attached during experiments. The final step of securing the needle is tightening the collet. Evans blue dye is used for visualization in E–H. Please click here to view a larger version of this figure.
7. Injections
NOTE: All instruments used in this procedure are sterilized before surgery and between each mouse.
Figure 4: Optimal size and orientation of the amniotic cavity for successful injections. (A) Uterine horn with multiple E7.5 deciduas, shaped like a string of spheres (bottom) compared to large intestine (top). (B) Grasp uterine tissue (white dotted lines) between deciduas. Avoid squeezing the deciduas (white arrowheads) directly with forceps as deciduas and developing embryos are fragile at this early stage and prone to resorption upon excessive external force. (C) Female in supine position with deciduas exposed in a Petri dish filled with PBS and mounted on four feet of modeling clay. The deciduas are stabilized by an additional piece of modeling clay, shaped like a cylinder. (D, E) Orientation of the AC is influenced by the side of the uterine horn that is exposed. If deciduas from the left uterine horn are used, the AC will face away from the clay stabilizer and will be easily accessible to the needle on the left (D). However, if the right uterine horn is used, the ectoplacental cone will instead face the needle, making it more difficult to access the AC (E). Therefore, when injecting into the right uterine horn, the clay stabilizer is placed towards the needle-facing side (F), and the entire heating table is rotated 180° (G). (H) Recommended injection volumes according to AC sizes. In general, cavities with a diameter ≤ 0.2 mm can be injected with a maximum of 69 nL. Diameters > 0.2 mm and ≤ 0.29 mm tolerate volumes up to 2 x 69 nL (138 nL) and cavities > 0.29 mm can be injected with 3 x 69 nL (207 nL). Scale bars = 1 mm. (I, J) Nanoinjector is attached to the rail system and can be moved in x- and/or z-planes. The angle of the needle can be adjusted with the inject angle wheel. (K, L) Needle tip (white arrowhead) is aligned with the AC when appearing brightest in the ultrasound (L). (M) Ultrasound image showing the injection process, in which the needle tip is in the AC and well aligned (white arrowhead). Abbreviations: A = Amnion; AC = Amniotic Cavity; ExC = Exocelomic Cavity; EC = Ectoplacental Cone. Please click here to view a larger version of this figure.
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., ≥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 × 1010 infectious units (IFU) achieve over 95% targeting, while titers of ~1 × 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'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).
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 µm. (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 µm. 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 µm. (G, H) Clonal targeting (G) and high-efficiency (H) targeting of hindbrain, magnified in (I, J). Scale bars = 200 µm. (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 µm. Abbreviations: NEPTUNE = neural plate targeting with in utero 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. Please click here to view a larger version of this figure.
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 µm. (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 µm. (D) Schematic and confocal image of E13.5 tongue mesenchyme after injections with fluorescent reporter virus at E7.5. Scale bar = 50 µm. Abbreviations: D = Dorsal; M = Mesenchyme; N = Nasopharynx; SLD = Sublingual Duct; SMD = Submandibular Duct; T = Tongue; V = Ventral; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
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 µm. (C) Boxed region of B showing the DRG with GFP expression in SOX2+ (white arrows) and NeuN+ (white arrowheads) cell populations. Scale bar = 20 µm. (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 µm. (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 µm. Abbreviations: NEPTUNE = neural plate targeting with in utero nano-injection; CNS = central nervous system; PNS = peripheral nervous system; DRGs = dorsal root ganglia; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
1 mL Syringe | BD Bioscience | 309628 | |
27 G Needle | BD Bioscience | 300635 | |
3.5 inches capillaries | Drummond Scientific | 3000203G/X | Were used to pull in house needles |
70 MHz MS Series transducer | Visual Sonics | MS700 | |
Aquasonic clear ultrasound gel | Parker Laboratories | Mar-50 | |
Autoclip Applier 9 mm | Angthos | 12020-09 | |
CD1 mice | Charles River, Germany | Crl:CD1(ICR) | Females: from age of 8 weeks old Males: from the age of 12 weeks old |
Cotton Swab | OneMed Sverige AB | 120788 | |
DPBS | Gibco | 14190094 | |
Dressing forceps delicate straight 13 cm | Agnthos | 08-032-130 | |
EG-400 Narishige Micropipette Grinder | Narishige | NA | |
EZ clips 9 mm | Angthos | 59027 | clips |
Iris Scissors, Super Cut, straight, 9 cm | Agnthos | 307-336-090 | |
Isofluorane | Baxter Medical AB | EAN: 50085412586613 | Purchased from Swedish Pharmacy |
Kimwipes | Kimberly Clarke | 7557 | |
Membrane Tape | Visual Sonics | SA-11053 | |
Micropipette Puller | Sutter Instrument | P-97 | |
Modeling Clay | Sense AB | 10209 | |
Mouse Handling Table | Visual Sonics | 50249 | |
Nanoject II Auto Injector Kit | Drummond | 3-000-205A | |
Parafilm | Bemis | HS234526C | |
Petri dish with central opening (low wall) | Visual Sonics | SA-11620 | |
Petri dish, (ØxH): 92 x 16 mm | Sarstedt | 82.1472.001 | |
Rely+On Virkon | DuPont | 130000132037 | disinfectant |
Silicone membrane | Visual Sonics | SA-11054 | |
Steri 250, hot bead sterilizer | Angthos | 31100 | |
Surgical Tape (1.25 cm x 9.14 m) | Medicarrier | 67034 | |
Vevo Compact Dual (Med. Air & O2) Anesthesia System | Visual Sonics | VS-12055 | |
Vevo Imaging Station 2 | Visual Sonics | VS-11983 | |
Vevo2100 | Visual Sonics | VS-20047 | |
Vicryl 6-0; C-3 needle, 45 cm purple filament | Agnthos | J384H |
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 >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 >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.
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 >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 >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.
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 >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 >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.