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...

<ol>
	<li>Loukogeorgakis, S., 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=In+utero+stem+cell+and+gene+therapy:+current+status+and+future+perspectives.">In utero stem cell and gene therapy: current status and future perspectives.</a> European Journal of Pediatric Surgery. 24, 237-245 (2014).</li><li>Vrecenak, J., 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=In+utero+hematopoietic+cell+transplantation:+recent+progress+and+the+potential+for+clinical+application.">In utero hematopoietic cell transplantation: recent progress and the potential for clinical application.</a> Cytotherapy. 15, 525-535 (2013).</li><li>Peranteau, W., 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=Correction+of+murine+hemoglobinopathies+by+prenatal+tolerance+induction+and+postnatal+nonmyeloablative+allogeneic+BM+transplants.">Correction of murine hemoglobinopathies by prenatal tolerance induction and postnatal nonmyeloablative allogeneic BM transplants.</a> Blood. 126 (10), 1245-1254 (2015).</li><li>Vrecenak, J., 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=Stable+Long-Term+Mixed+Chimerism+Achieved+in+a+Canine+Model+of+Allogeneic+in+utero+Hematopoietic+Cell+Transplantation.">Stable Long-Term Mixed Chimerism Achieved in a Canine Model of Allogeneic in utero Hematopoietic Cell Transplantation.</a> Blood. 124 (12), 1987-1995 (2014).</li><li>Peranteau, W., 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=CD26+Inhibition+Enhances+Allogeneic+Donor-Cell+Homing+and+Engraftment+after+in+utero+Hematopoietic-Cell+Transplantation.">CD26 Inhibition Enhances Allogeneic Donor-Cell Homing and Engraftment after in utero Hematopoietic-Cell Transplantation.</a> Blood. 108 (13), 4268-4274 (2006).</li><li>Waddington, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+gene+transfer+of+human+factor+IX+to+fetal+mice+can+induce+postnatal+tolerance+of+the+exogenous+clotting+factor.">In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor.</a> Blood. 101 (4), 1359-1366 (2003).</li><li>Stitelman, 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=Developmental+Stage+Determines+Efficiency+of+Gene+Transfer+to+Muscle+Satellite+Cells+by+in+utero+Delivery+of+Adeno-Associated+Virus+Vector+Serotype+2/9.">Developmental Stage Determines Efficiency of Gene Transfer to Muscle Satellite Cells by in utero Delivery of Adeno-Associated Virus Vector Serotype 2/9.</a> Molecular Therapy - Methods &amp; Clinical Development. 1, 14040 (2014).</li><li>Endo, 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=Gene+Transfer+to+Ocular+Stem+Cells+by+Early+Gestational+Intraamniotic+Injection+of+Lentiviral+Vector.">Gene Transfer to Ocular Stem Cells by Early Gestational Intraamniotic Injection of Lentiviral Vector.</a> Molecular Therapy. 15 (3), 579-587 (2007).</li><li>Boelig, 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=The+Intravenous+Route+of+Injection+Optimizes+Engraftment+and+Survival+in+the+Murine+Model+of+In+utero+Hematopoietic+Cell+Transplantation.">The Intravenous Route of Injection Optimizes Engraftment and Survival in the Murine Model of In utero Hematopoietic Cell Transplantation.</a> Biology of Blood and Marrow Transplantation. 22 (6), 991-999 (2016).</li><li>Wu, C., 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=Intra-amniotic+Transient+Transduction+of+the+Periderm+with+a+Viral+Vector+Encoding+TGF&#946;3+Prevents+Cleft+Palate+in+Tgf&#946;3-/-.+Mouse+Embryos.">Intra-amniotic Transient Transduction of the Periderm with a Viral Vector Encoding TGF&#946;3 Prevents Cleft Palate in Tgf&#946;3-/-. 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>

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 t...

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.

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			<td style="width:168px;">Medline</td>
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			<td height="21" style="height:21px;width:281px;">Vertical Micropipette Puller</td>
			<td style="width:168px;">Sutter Instruments</td>
			<td style="width:147px;">P-30</td>
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			<td style="width:168px;">Sutter Instruments</td>
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			<td height="21" style="height:21px;width:281px;">IM-300 Pneumatic Microinjector</td>
			<td style="width:168px;">Narishige</td>
			<td style="width:147px;">IM-300</td>
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			<td height="21" style="height:21px;width:281px;">Insulin Syringe&#160;</td>
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			<td style="width:147px;">305935</td>
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			<td height="21" style="height:21px;width:281px;">Filter</td>
			<td style="width:168px;">Genesee Scientific</td>
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			<td height="21" style="height:21px;width:281px;">Compac5 Anesthesia Machine</td>
			<td style="width:168px;">VetEquip Compac5</td>
			<td style="width:147px;">901812&#160;</td>
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			<td height="21" style="height:21px;width:281px;">Isoflurane</td>
			<td style="width:168px;">Piramal Critical Care</td>
			<td style="width:147px;">NDC 66794-017-25</td>
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			<td height="21" style="height:21px;width:281px;">N2 gas</td>
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			<td height="21" style="height:21px;width:281px;">Ad-GFP viral vector</td>
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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 o...

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 ...

This method can help answer key questions in embryology, gene therapy and stem cell transplantation. An advantage to this technique is that it enables analysis of the effects of different experimental therapies on Murine fetuses. Demonstrating this procedure will be doctors Nicholas Ahn, and Barbara Coons, physicians and research fellows in our laboratory.Before beginning the procedure, drop five to 10 microliters of sterile PBS into and out of the surgical needle tip, two to three times to clear it of any possible debris. Next fill the needle with the reagent of interest at the appropriate experimental volume and concentration. And press the mode button three times on the micro injector to advance to the injection calibration screen.Add intervals of 10 or 100 milliseconds to adjust the injection time, and press the mode button two more times. Press the balance button and push the foot pedal once. Then press the pedal again and assess what volume of solution is emptied out of the needle per push.When the micro injector is calibrated to the appropriate experimental volume, fill the needle to the correct level for the injection. When all of the needles have been cleaned, confirm a lack of response to toe pinch of the pregnant two to six month old female mouse. Shave the abdomen, taking care not to damage the nipples, and transfer the animal to a heating pad in the supine position.Apply ointment to the animal's eyes and secure the limbs to the heating pad with tape. And disinfect the exposed skin. Then inject local anesthetics such as 100 microliters of 0.25%Bupivacaine subcutaneously.Next, make a one to two centimeter skin incision such that the lower border is no closer than one centimeter to the introitus, and identify the midline of the fascia, taking care not to injure the surrounding epigastric vessels. Using Adson forceps, pinch the fascia without grabbing any of the underlying organs or fetuses, and open the tissue with scissors. Once safely within the abdominal cavity, extend the fascial incision to the length of the skin incision, and use cotton tip applicators to move the intestines into the upper part of the abdomen to expose the gravid uterus.Extract the uterus through the incision carefully identifying the right and left ovaries to ensure that all of the fetuses are counted. Place the left uterus back into the abdomen so that only the right uterus is exposed, to keep the non injected fetuses warm. And gently grasp at the most lateral amniotic sac between the thumb and index fingers of the non dominant hand.Position the dissection microscope over the animal and adjust the focus and lighting for a better visualization of the fetus as necessary. For an intravenous injection rotate the uterus so that the vitelline vain, to be injected is parallel to the needle tip. And lay the needle on the uterus at a five degree angle to pierce the uterine wall.With the tip between the uterine wall and the amniotic sac, place the tip directly atop the vitelline vain and at a nearly tangential angle, glide the needle over the vain until the bevel advances into the vessel. It's imperative that the angle stays low so that the needle does not penetrate the back wall of the vessel. When a spot of blood is observed at the needle tip, to press the foot pedal to inject the full volume of the material of interest into the vessel.For an intra amniotic injection, rotate the amniotic sac until a location devoid of vessels is found. Point the needle perpendicular to the uterine wall and pierce the needle through the uterus yolk sac, and amniotic sac taking care not penetrate any fetal tissue. Then inject the appropriate volume of material desired.After either type of injection, withdraw the needle from the injection site once the desired volume is delivered. And proceed to the next fetus until all of the fetuses of the right uterine horn have been injected. Then return the right uterine horn into the abdomen.And remove the left uterine horn out of the abdomen. When all the fetuses in the left horn have been injected return the entire uterus to the abdomen, taking care to avoid a uterine or intestinal volvulus. Use a disposable transfer pipette to dispense about two milliliters of sterile PBS into the abdomen, to replace any insensible losses.And use 4-0 polyglactin 910 sutures to close the fascia and abdomen in one continuous layer. Then transfer the mouse to a clean cage under a heat lamp with monitoring until full recumbency. In this training experiment the livers of the fetuses that received in-utero transplantation by a trainee displayed lower fluorescent levels 24 hours after injection, due to a lower engraftment of the transplanted GFP cells, than did livers injected by an experienced instructor.Flow cytometric analysis for GFP positive donor cells also correlated with the lower level of fluorescents observed under fluorescent microscopy. 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 hours following the in utero injection of Adeno virus carrying the GFP transgene in gestational day 12.5 fetuses.Once mastered this technique can completed in 15-25 minutes per pregnant mouse. After watching this video you should have a good understanding of how to safely and effectively deliver different therapies to mouse fetuses.

intravenous-intra-amniotic-utero-transplantation-murine

Research

JoVE Journal

Developmental Biology

9.3K visualizações.  Children's Hospital of Philadelphia. Este método pode ajudar a responder a perguntas-chave em embriologia, terapia genética e transplante de células-tronco. Uma vantagem para esta técnica é que permite a análise dos efeitos de diferentes terapias experimentais em fetos murinos. Demonstrando este procedimento estarão os médicos Nicholas Ahn, e Barbara Coons, médicos e pesquisadores em nosso laboratório.Antes de iniciar o procedimento, solte de cinco a dez microlitadores de PBS estéreis dentro e fora da ponta da ...