The experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children&#39;s Hospital of Philadelphia.
1. Creation of Injection Pipettes
<ol>
	<li>Using a vertical micropipette puller, pull a 100 &#181;L microcapillary pipette (Figure&#160;1A - 1C). Calibrate the micropipette puller so that the tapered end is &#62; 1 cm long. 
	NOTE: Initially, the settings of the puller should be adjusted for an optimum length. A higher heat setting will make the tip longer, and a higher pull setting will make the diameter of the tip narrower.</li>
	<li>Cut the tapered end so that it is &#8805; 1 cm long. Ensure that the internal diameter at the tip of the needle is between 70 &#181;m and 100 &#181;m and that it is inversely proportional to the length of the tapered end. 
	NOTE: The internal diameter also depends on the calibration of the micropipette puller. See the instructions from the manufacturer of the vertical micropipette puller or any preferred micropipette puller type.</li>
	<li>After ensuring that the needle has the correct internal diameter, create the bevel of 15 - 20 degrees by sharpening the tip using a micropipette beveller with a diamond sharpening wheel (Figure&#160;2A - 2C). Make sure to rest the tip gently on the wheel without too much pressure, to decrease chances of breaking or cracking the tip. 
	NOTE: A paintbrush can be used to wipe away any debris that builds up on the needle tip.</li>
	<li>Evaluate the tip under a microscope and ensure that the tip is round without any chips or cracks. Reevaluate the internal diameter to make sure it is between 70 &#181;m and 100 &#181;m (Figure&#160;2D - 2H).</li>
	<li>Draw lines on the rest of the needle to designate 5 &#181;L of volume between them (e.g., needles with an internal diameter of 1.3 mm should have lines drawn at 3.77 mm increments).</li>
	<li>Autoclave the needles prior to use in surgery and handle them with sterile gloves.&#160;</li>
</ol>
2. In utero Injections
<ol>
	<li>Prepare the necessary instruments by autoclaving them ahead of time. Include essential instruments such as microinjector needle holder, a surgical needle driver, a pair of Adson forceps, a pair of curved regular tissue scissors, a 1 mL insulin syringe, a couple of cotton-tip applicators, a transfer pipette, a 50 mL conical tube, and a pack of 4-0 polyglactin 910 sutures.</li>
	<li>Using a sterile technique, attach the needle to the needle holder and plug it into the microinjector. 
	NOTE: The settings of the compressed nitrogen used are as followed: inject 4 - 6 psi, balance 0 psi. Depending on what is being injected, specifically, the viscosity of the injectate, as well as the size of the micropipette, the injection times vary between 0.3 - 1.5 s.</li>
	<li>Clean out the needle tip of any possible debris by drawing up 5 - 10 &#181;L of sterile 1x phosphate-buffered saline (PBS) and then clearing it out. Repeat this 2 - 3x.</li>
	<li>Prepare pregnant 2- to 6-month-old female mice for surgery by shaving their abdomens with a clipper. Be careful not to damage the nipples. Administer oral pain medication (e.g., 100 &#181;L of 1.5 mg/mL meloxicam oral suspension per mouse).</li>
	<li>Start filling the needle with the desired material (cells/vector/drug) at the desired volume. Be careful not to break the needle tip while filling the needle. 
	NOTE: The volume of injection per fetus varies depending on the specific experimental design. 20 &#181;L works well for injecting a large number of cells (i.e., up to 107 cells into the vitelline vein. For instance, we delivered 1 x 107 whole bone marrow cells isolated from C57BL/6TgN(act-EGFP)OsbY01 [&#34;B6 Green Fluorescent Protein (GFP)&#34;] mice via the vitelline vein into gestational day-14 Balb/c fetuses. For viral vector injections, a single injection of 10 &#181;L of a 1:1 diluted vector with PBS works well.</li>
	<li>To calibrate the injection time, take the following steps.
	<ol>
		<li>Push the Mode button 3x on the microinjector to get to the injection calibration screen. Adjust the injection time by adding intervals of 10 or 100 ms and push the Mode button 2x.</li>
		<li>Push the Balance button and push the foot pedal once. Now push the pedal again and assess how much volume is emptied out of the needle per push. If not calibrated to the desired volume of 5 - 20 &#181;L per pedal push, repeat steps 2.6.1 and 2.6.2. 
		NOTE: Generally, it is good to calibrate each push to deliver half the total target volume at a time. While it is possible to inject more than 30 &#181;L or even 40 &#181;L of the total volume, we do not generally go over 20 &#181;L per fetus, intravenous or intra-amniotic.</li>
	</ol>
	</li>
	<li>Fill the needle up to the desired level.</li>
	<li>Start delivering anesthesia to the mouse by adjusting the oxygen flowmeter to 1 L/min and the isoflurane vaporizer to 3%.</li>
	<li>Confirm whether the mouse is anesthetized by checking for the absence of the pedal reflex. Transfer the mouse to a heating pad in a supine position.</li>
	<li>Apply lubricant eye gel to avoid corneal desiccation. Secure the mouse in place by taping the upper and lower limbs to the pad.</li>
	<li>Prep the abdomen with chlorhexidine scrub followed by alcohol&#160;and inject a local anesthetic (e.g., 100 &#181;L of 0.25% bupivacaine) subcutaneously (Figure 3A).</li>
	<li>With scissors, make a 1 - 2 cm skin incision so that the lower border is no closer than 1 cm to the introitus; the fascia beneath is very thin and translucent.</li>
	<li>Identify the midline of the fascia which is more transparent than the surrounding area. Be careful not to injure the epigastric vessels which lie on either side of the midline. Should the epigastric vessels get injured, hold pressure with cotton-tip applicators to stop the bleeding.</li>
	<li>Using Adson forceps, pinch the fascia without grabbing any of the underlying organs such as the intestines, bladder, or fetuses. Open the fascia with scissors, being careful not to damage any of the organs. Once safely in the abdomen, extend the fascial incision. Make it no longer than the skin incision.</li>
	<li>Use cotton-tip applicators to move the intestines into the upper part of the abdomen, thus exposing the gravid uterus. Deliver the uterus out of the incision, carefully identifying the right and left ovaries to ensure all fetuses are counted (Figure 3B).</li>
	<li>Place the left uterus back into the abdomen so that only the right uterus is exposed; this prevents the desiccation of the uterus and keeps the non-injected fetuses warm.</li>
	<li>Hold the most lateral fetus/amniotic sac between the thumb and index fingers of the operator&#39;s non-dominant hand (Figure 3C). Always be gentle as to not cause any damage to the fetuses.</li>
	<li>Position the dissection microscope (a 10X magnification is ideal) and adjust the focus so that the fetus is in view. Adjust the lighting for a better visualization.</li>
	<li>Identify the part that will be injected (vitelline vein, amnion).For intravenous injections, visualize both the vitelline veins and their anastomosis first. For intra-amniotic injections, orient the fetus with the right side in view.</li>
	<li>Reach the target space with the needle as described below.
	<ol>
		<li>For an intravenous injection, do as follows.
		<ol>
			<li>Rotate the uterus so that the vitelline vein that is being injected is parallel to the tip of the needle; keep in mind that the injections must be made towards the anastomosis of the two veins.</li>
			<li>Lay the needle on the uterus at a 5&#176; angle and pierce the uterine wall. Now that the tip is between the uterine wall and the amniotic sac, place the tip directly atop the vitelline vein.</li>
			<li>At a nearly tangential angle, glide the needle over the vein until the bevel pierces and advances into the vessel; this is evident by a flash of blood seen in the needle tip (Figure 3D). 
			NOTE: Accessing the vein may take a few tries as the needle may not pierce the vein with the first glide over the vein.</li>
		</ol>
		</li>
		<li>For an intra-amniotic injection, do as follows.
		<ol>
			<li>Rotate the amniotic sac and find a location devoid of vessels to pierce.</li>
			<li>Point the needle perpendicular to the uterine wall and pierce through the uterus, the yolk sac, and then the amniotic sac. Be careful not to pierce through any fetal tissue. Make sure the needle has passed between the limbs as this confirms that the needle is in the amniotic sac. Then proceed with the injection.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Inject the appropriate volume of material desired (usually 10 - 20 &#181;L) by pushing the foot pedal. 
	NOTE: Because the injector must maintain the visualization of the needle tip through the microscope at all times, a second person must read the markings on the needle to quantify the injected amount and inform the injector how much volume is left to inject. A discussion prior to the injection between the injector and assistant is important to avoid any confusion and delay. This is especially important for intravenous injections because a delayed removal of the needle will allow a backflow of venous blood into the needle and result in an inaccurate dosing.</li>
	<li>Withdraw the needle from the injection site once the desired volume is delivered. As there may be some bleeding from the vessel puncture site with intravenous injections, hold pressure with the side of the needle for 10 - 15 s to stop the bleeding.</li>
	<li>Proceed to the next fetus. Continue until all the fetuses of the right uterine horn have been injected.</li>
	<li>Remove the left uterine horn from the abdomen and replace the right uterine horn back inside the peritoneal cavity. 
	NOTE: Occasionally, the needle needs to be refilled with the injectant.</li>
	<li>Once all fetuses have been injected, replace the uterus into the abdomen (Figure 3E). Make sure to avoid a uterine or intestinal volvulus.</li>
	<li>With a disposable transfer pipette, place roughly 2 mL of 1x PBS into the abdomen to replace any insensible losses.</li>
	<li>Close the fascia and abdomen in one continuous layer using 4-0 polyglactin 910 sutures to avoid injuring the underlying organs during the closure (Figure&#160;3F - 3G).</li>
	<li>Remove the tape and transfer the mouse to a cage underneath a heat lamp. Be careful not to place the heat lamp too close to the mouse. Make sure the cage has bedding, food, and water. 
	NOTE: A thermostatically controlled warm chamber may also be used. The mouse is awake when it is upright and walking.</li>
	<li>Observe the mouse daily and give pain medication as needed. 
	NOTE: We routinely give meloxicam on postoperative days 1 and 2, and sometimes on day 3 if the mouse shows signs of pain.</li>
	<li>If doing a batch surgery with the same injection material, clean out the injection needle with sterile PBS. If injecting with a different material, dispose of the needle in a sharps container and use a new needle. 
	NOTE: We recommend fostering the pups with surrogate dams immediately after birth in case the dam mounts an immune response to the injectant and transfers antibodies to the pups via breast milk.</li>
</ol>

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Mouse Embryos.</a> Molecular Therapy. 1, 8-17 (2013).</li><li>Roybal, J., Endo, M., Radu, A., Zoltick, P., Flake, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+gestational+gene+transfer+of+IL-10+by+systemic+administration+of+lentiviral+vector+can+prevent+arthritis+in+a+murine+model.">Early gestational gene transfer of IL-10 by systemic administration of lentiviral vector can prevent arthritis in a murine model.</a> Gene Therapy. 18 (7), 719-726 (2011).</li><li>Reay, 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=Full-Length+Dystrophin+Gene+Transfer+to+the+Mdx+Mouse+in+utero.">Full-Length Dystrophin Gene Transfer to the Mdx Mouse in utero.</a> Gene Therapy. 15 (7), 531-536 (2008).</li><li>Ahmed, S., Waddington, S., Boza-Mor&#225;n, M., Y&#225;&#241;ez-Mu&#241;oz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Efficiency+Transduction+of+Spinal+Cord+Motor+Neurons+by+Intrauterine+Delivery+of+Integration-Deficient+Lentiviral+Vectors.">High-Efficiency Transduction of Spinal Cord Motor Neurons by Intrauterine Delivery of Integration-Deficient Lentiviral Vectors.</a> Journal of Controlled Release. 273, 99-107 (2018).</li><li>Haddad, M., Donsante, A., Zerfas, P., Kaler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+Brain-Directed+AAV+Gene+Therapy+Results+in+Rapid,+Robust,+and+Persistent+Transduction+of+Mouse+Choroid+Plexus+Epithelia.">Fetal Brain-Directed AAV Gene Therapy Results in Rapid, Robust, and Persistent Transduction of Mouse Choroid Plexus Epithelia.</a> Molecular Therapy - Nucleic Acids. 2, 101 (2013).</li><li>Nijagal, A., Le, T., Wegorzewska, M., MacKenzie, T. <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> Journal of Visualized Experiments. (47), e2303 (2011).</li><li>Davey, M., 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=Jaagsiekte+Sheep+Retrovirus+Pseudotyped+Lentiviral+Vector-Mediated+Gene+Transfer+to+Fetal+Ovine+Lung.">Jaagsiekte Sheep Retrovirus Pseudotyped Lentiviral Vector-Mediated Gene Transfer to Fetal Ovine Lung.</a> Gene Therapy. 19 (2), 201-209 (2011).</li></ol>

The authors have nothing to disclose.

In utero transplantation is a potential therapy for many congenital disorders that can be diagnosed early in gestation. The murine model for IUT allows researchers to explore the fetal environment or to experiment with different therapies. Depending on what is being injected and what is being targeted, intravenous or intra-amniotic in utero transplantation can provide a reliable delivery of an injectant into the desired space.
When targeting specific organs, it is important to pick the appropriate embryological age of the fetus as well as the injection technique. While the intravenous injection of cells at E14 is ideal for targeting the hematologic niche, and the intra-amniotic injection at E16 is ideal for lung targeting, these are not the only options available. For example, intra-amniotic injections can be performed for fetuses as early as E8 with ultrasound guidance8. Systemic delivery is also possible before E14 with ultrasound-guided intra-cardiac injections at E9 - E1011. The feasibility of performing injections at various stages of the fetus&#39; development offers great potential for experiments investigating the safety and efficiency of gene transfers and cell transplants as well as investigating basic questions of developmental biology.
Furthermore, in addition to an intravenous and intra-amniotic delivery, other sites are also available for targeting depending on the purpose of the therapy or the scientific question being pursued. The in utero intramuscular approach has been used for a gene transfer for muscular dystrophies12, an intraspinal approach for the transduction of spinal cord motor neurons13, and an intracranial approach for gene transfers to target central nervous system diseases14. For in utero hematopoietic cell transplantation, intrahepatic and intraperitoneal routes are additional viable options as each route of delivery ultimately targets the hematopoietic niche15. However, the intravenous transplantation route allows for a more efficient homing of the donor cells into the hematopoietic niche and a larger dosing of the donor cells, thus resulting in overall higher levels of stable long-term donor cell engraftment without added fetal mortality1.
The protocols we have detailed above for performing IUT are powerful and versatile tools that allow for a unique in vivo approach to studying stem cell biology, developmental immunology and immunologic tolerance induction, developmental biology, and prenatal gene therapy/genome editing. These delivery methods also have relevant clinical implications and have been the basis for studies of IUHCT and in utero gene therapy in pre-clinical large animal models such as the canine and the ovine model4,16. They will continue to serve as a valuable tool to test new ideas in developmental biology and explore new therapies for devastating congenital genetic, hematologic, immune, and metabolic disorders.

Recent advances in antenatal screening and diagnosis have brought to light the possibility of treating the fetus for a number of congenital disorders which do not have adequate postnatal treatment options and result in significant morbidity and mortality. Specifically, in utero hematopoietic stem cell transplantation (IUHCT) and gene therapy/genome editing have the potential to take advantage of normal developmental properties of the fetus to treat congenital hematologic, immune, and genetic disorders more efficiently than postnatal HSC transplantation and gene therapy/genome editing can do1,2. Specifically, due to the small size of the fetus, the donor cell or viral vector dose can be maximized per the weight of the recipient. Additionally, the immunologic immaturity of the fetus allows donor HSCs to be injected without the myeloablative and immunosuppressive conditioning that is required in postnatal transplant protocols. Similarly, viral vectors carrying a therapeutic transgene or genome editing technology can be injected without a limiting immune response to either the transgene product or the viral vector. Finally, the accessibility and proliferative nature of fetal stem/progenitor cells afford the possibility of a more efficient transduction of target progenitor cells, as well as certain modes of genome editing (homology-directed repair) which require cycling cells to occur efficiently. The murine model serves as an insightful and affordable means to address important questions in stem cell biology and immunology prior to experimenting in pre-clinical large animal models and, as such, has served as the primary model in which IUHCT and in utero gene therapy have been explored1,2,3.
Although many variables play an important role in the success of IUHCT and in utero gene therapy/genome editing in murine and large animal models, a key variable is the method of delivery of the HSCs or viral vector. The delivery of large doses of donor HSCs with a first-pass effect occurring in the fetal liver, the hematopoietic organ at the time of the IUHCT, has been shown to be instrumental in achieving macrochimeric levels of engraftment in mouse and large animal models4,5. This was achieved via an injection of donor cells via the vitelline vein in the mouse model and via an intra-cardiac injection in the canine model. The route of injection also plays a fundamental role in targeting progenitor cells of different organs during development. For example, an intravenous injection via the vitelline vein has been shown to efficiently transduce cardiomyocytes and hepatocytes following a late gestation injection6,7. Alternatively, an intra-amniotic injection of viral vectors allows the targeting of organs that are physically exposed based on the embryonic folding/development at the time of the injection8. This is best exemplified by the targeting of respiratory epithelium via an intra-amniotic injection late in the gestation to take advantage of normal fetal &#34;breathing&#34; movements, which exposes the respiratory tract to the viral vector in the amniotic fluid9. These two modes of IUT, intravenous via the vitelline vein and intra-amniotic, have been the basis for multiple past and ongoing experiments in our laboratory. In this protocol, we describe in detail the methods for performing intravenous and intra-amniotic IUT in the murine model.

<table><tbody><tr><td>Gloves</td><td>Cardinal Health</td><td>2D73DP65</td><td></td></tr><tr><td>Adson Forceps w/ teeth</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Adson Forceps w/o teeth</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Curved scissors</td><td>Fine Science Tools</td><td>14075-11</td><td></td></tr><tr><td>Heavy Scissors</td><td>Fine Science Tools</td><td>14002-13</td><td></td></tr><tr><td>Needle Driver</td><td>Fine Science Tools</td><td>12005-15</td><td></td></tr><tr><td>Vicryl 2.0</td><td>Ethicon</td><td>JB945</td><td></td></tr><tr><td>Transfer Pipette</td><td>Medline</td><td>GSI135010</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>Medline</td><td>MDS202000</td><td></td></tr><tr><td>50 mL Conical tube</td><td>Fischer Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>Eye lubricant</td><td>Major LubriFresh</td><td>0904-6488</td><td></td></tr><tr><td>Heating Pad</td><td>K&amp;amp;H</td><td>3060</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ16</td><td></td></tr><tr><td>Injector</td><td>Narishige</td><td>HI01PK01</td><td></td></tr><tr><td>Glass Capillary tubes</td><td>Kimble</td><td>71900-100</td><td></td></tr><tr><td>Vertical Micropipette Puller</td><td>Sutter Instruments</td><td>P-30</td><td></td></tr><tr><td>Microelectrode Beveler</td><td>Sutter Instruments</td><td>BV-10</td><td></td></tr><tr><td>IM-300 Pneumatic Microinjector</td><td>Narishige</td><td>IM-300</td><td></td></tr><tr><td>Insulin Syringe&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>305935</td><td></td></tr><tr><td>Filter</td><td>Genesee Scientific</td><td>25-244</td><td></td></tr><tr><td>Compac5 Anesthesia Machine</td><td>VetEquip Compac5</td><td>901812&amp;nbsp;</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>N2 gas</td><td>Airgas</td><td>NI 125</td><td></td></tr><tr><td>O2 gas</td><td>Airgas</td><td>OX 125</td><td></td></tr><tr><td>Ad-GFP viral vector</td><td>Penn Vector Core</td><td>H5&#039;.040.CMV.eGFP</td><td></td></tr></tbody></table>

intravenous and intra-amniotic in utero transplantation in the murine model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Survival and engraftment are important measures of success for IUHCT experiments. Depending on the specific endpoints of an experiment, fetuses that received an IUHCT may be analyzed prenatally by a C-section or postnatally. On average, the survival rates after intravenous injections range from 75 - 100%. The survival rates after intra-amniotic injections tend to fair better than intravenous injections, at around 85 - 100%.
In our laboratory, the training process to attain proficiency in these techniques takes approximately 8 - 12 months. To assess the acquisition of the skills required to perform these injections in a reproducible fashion, trainees are monitored for fetal survival and donor cell engraftment at short time points following the IUT. This is demonstrated by the following quality control experiments. Specifically, 1 x 107 whole bone marrow cells are isolated from C57BL/6TgN(act-EGFP)OsbY01 (&#34;B6 GFP&#34;) mice as previously described5 and are injected via the vitelline vein into gestational day-14 Balb/c fetuses. In one group, the IUT was performed by an experienced instructor, and in the other group by a trainee who has been practicing the technique for ~ 4 months. Representative fluorescent microscopy images of the harvested fetal livers 24 h after the IUT are shown in Figure 4A. The livers of the fetuses who received the IUT by the trainee fluoresce less due to a lower engraftment of the transplanted GFP cells. These livers were then analyzed for GFP+ donor cells by flow cytometry to quantify the engraftment levels. The difference in mean engraftment levels between the two injectors correlates with the images seen under fluorescent microscopy (Figure 4B).
The ability of viral vectors delivered via the intra-amniotic route to transduce cells in contact with the amniotic fluid is exemplified by the transduction of epithelial cells 48 h following an intra-amniotic injection of Ad-GFP (an adenovirus vector carrying GFP transgene10) in gestational day-12.5 fetuses (Figure&#160;5A and 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58047/58047fig1.jpg" /> 
Figure 1. The process of making a glass pipette needle with the micropipette puller. (A) Mount the glass pipette and secure it by tightening the dials on either end of the puller. (B) Once secured, the pulling switch activates the heat. The settings shown are Heat #1: 985 and Pull: 27. The dark cover glass should be closed during this process for safety. It was opened for photoshoot purposes. (C) The micropipette is separated. Discard the bottom portion and take the top portion of the separated micropipette for the next steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58047/58047fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58047/58047fig2.jpg" /> 
Figure 2. The process of grinding the micropipette to shape the tip of the needle. (A) This panel shows the general setup of the grinding. A light source is needed to visualize the tip under the microscope. (B and C) Mount the micropipette at a 15 - 20&#176; angle. (D) A buildup of debris is expected throughout the grinding process. Use a paintbrush to clear the tip to get a better visualization of the grinding process. (E) A well-ground needle tip without any identifiable chips or jagged edges is shown under a 4X magnification. (F, G, and H) A re-inspection of the ground needle under a 10X magnification shows a well-ground needle with a sharp tip and smooth edges around the tip. Make sure to check the needle at different angles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58047/58047fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58047/58047fig3.jpg" /> 
Figure 3. Laparotomy and in utero transplantation. (A) Shave, anesthetize, and tape a pregnant dam and prep her abdomen. (B) After the laparotomy, deliver the entirety of the uterus outside the abdomen for an identification of all fetuses. (C) Position the fetus with the surgeon&#39;s non-dominant index finger and thumb while maintaining tension with the third finger. Identify the tip of the needle under the microscope in relation to the fetus. (D) A flash of blood back-flowing into the micropipette needle must be seen upon the cannulation of the vitelline vein. (E) After a completion of all injections, place all the fetuses back into the abdomen. (F) Close the abdomen with a single-layer running stitch using 4-0 polygalactin 910 sutures. (G) Once the abdomen is fully closed, let the dam recover under a heat lamp. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58047/58047fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58047/58047fig4.jpg" /> 
Figure 4. Engraftment of donor whole bone marrow mononuclear cells after a vitelline vein injection. (A) The difference in the degree of engraftment with (trainee) and without (instructor) the leakage of cells is shown clearly under fluorescent microscopy. (B) Percent chimerism also reflected the same finding shown by the flow cytometry analysis. Each data point represents the liver from a different injected fetus. The experiment was performed by one trainee and one instructor. The error bars represent a standard deviation (SD). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58047/58047fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58047/58047fig5.jpg" /> 
Figure 5. The expression pattern of green fluorescent protein in a fetal embryo 48 h after an intra-amniotic injection of Ad-GFP. (A) This panel shows the cornea (red arrow) and skin stained with GFP at E14.5 after an intra-amniotic injection of Ad-GFP at E12.5 (E12.5/E14.5). (B) A cryosection of the back of the embryo (indicated with a light blue box in panel A) at a higher magnification shows that the viral transduction is limited to the superficial peridermal cell layer (red arrows) and not epidermis (epi). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58047/58047fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model at JoVE.com

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

intravenous-intra-amniotic-utero-transplantation-murine

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.5K Views.  Children's Hospital of Philadelphia. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in embryologie, gentherapie en stamceltransplantatie. Een voordeel van deze techniek is dat het analyse van de effecten van verschillende experimentele therapieën op Murine foetussen mogelijk maakt. Het aantonen van deze procedure zal artsen Nicholas Ahn, en Barbara Coons, artsen en onderzoekers in ons laboratorium.Voordat u met de procedure begint, laat u vijf tot 10 microliter steriele PBS in en uit de chirurgische ...

Video: Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Medicine

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 Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat 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 rats 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 E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

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

Biology

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 ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

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.

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.

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

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer from AUFI. These women were deprived of the option to have children until the advent of UTx. The clinical application of UTx was driven by experimental studies in animals, and the first successful UTx was achieved in rats. Given their physiological, immunological, genetic, and reproductive characteristics, rats are a suitable model system for such transplants. In particular, their short gestation period is a clear advantage, as the usual endpoint of experimental UTx is successful pregnancy with live birth. The biggest challenge for rat models remains the small anatomy, which requires advanced microsurgical skills and experience. Although UTx has led to pregnancy in the clinic, the procedure is not established and requires continuous experimental optimization. Here, a detailed protocol is presented, including essential troubleshooting for rat UTx, which is expected to make the entire procedure easier to grasp for those without experience in this type of microsurgery.

The present protocol describes all the essential steps for successful uterine transplantation (UTx) in rats. The rat model has proven suitable to promote the clinical implementation of UTx; however, rat UTx is a highly complex procedure requiring careful instructions.

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

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