Name: intravenous and intra-amniotic in utero transplantation in the murine model
Uploaded: 2018-10-09T13:00:00
Description: Watch this Scientific Journal Video about Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model at JoVE.com

实验性的议定书得到了费城儿童医院的机构动物护理和使用委员会的批准。
1. 注射吸管的创建
<ol><li>使用垂直微拉拔器, 拉出100µL microcapillary 吸管 (图 1A -1C)。校准微拉拔器, 使锥形端 > 1 厘米长。 注意: 最初, 拉拔器的设置应调整为最佳长度。较高的热设置将使尖端更长, 更高的拉动设置将使尖端的直径更窄。</li>
	<li>切锥形端, 使它是≥...

<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>

在子宫移植是一个潜在的治疗许多先天性疾病, 可以诊断早期在妊娠期。IUT 的小鼠模型允许研究人员探索胎儿环境或尝试不同的治疗方法。根据正在注射的东西和被靶向的,在子宫内移植中静脉注射或羊膜腔可以提供可靠的 injectant 到所需的空间。
当靶向特定器官时, 选择合适的胎儿胚胎年龄以及注射技术是很重要的。虽然静脉注射细胞在 E14 是理想的靶向血液的?...

最近在产前筛查和诊断方面的进展, 使人有可能治疗胎儿的一些先天性疾病, 没有足够的产后治疗选择, 并导致严重的发病率和死亡率。具体地说,在子宫内造血干细胞移植 (IUHCT) 和基因治疗/基因组编辑有潜力利用胎儿正常发育特性, 以治疗先天性血液, 免疫和遗传障碍比产后 HSC 移植和基因治疗/基因组编辑可以做1,2。具体来说, 由于胎儿的体积小, 捐献者细胞或病毒载体剂量可以被最大化的每一个受体的重量。此外, 胎儿的免疫不成熟允许捐献者 HSCs 注射, 而不需要清髓性和免疫抑制的条件下, 产后移植协议。同样, 携带治疗性转基因或基因组编辑技术的病毒载体也可以在不受转基因产品或病毒载体的限制免疫反应的情况下注射。最后, 胎儿茎/祖细胞的可得性和增殖性为目标祖细胞提供了更有效的转导的可能性, 以及基因组编辑的某些模式 (同源定向修复), 需要循环细胞有效地发生。小鼠模型是一个有洞察力和负担得起的方法, 以解决干细胞生物学和免疫学的重要问题之前, 实验前的大型动物模型, 并因此, 作为一个主要的模式, 其中 IUHCT 和在子宫内基因治疗已经探索了1,2,3。
虽然许多变量在 IUHCT 的成功和在小鼠和大动物模型中的子宫基因治疗/基因组编辑中起着重要作用, 但关键变量是 HSCs 或病毒载体的传递方法。大剂量的捐献者 HSCs 在胎儿肝脏 (IUHCT 时的造血器官) 中发生的第一次效应, 已被证明有助于在小鼠和大型动物模型中实现植入 macrochimeric 水平4 ,5。这是通过在老鼠模型中通过卵黄静脉注入供体细胞和通过心脏内注射的犬模型来实现的。注射途径在发育过程中对不同器官的祖细胞有重要的靶向作用。例如,通过卵黄静脉静脉注射已证明有效地传感器心肌细胞和肝细胞后, 后期妊娠注射6,7。另外, 羊膜内注射病毒载体允许靶向身体暴露的器官根据胚胎折叠/发展在注射8的时候。这是最好的例证, 通过羊膜腔内注射在妊娠晚期, 以利用正常的胎儿 "呼吸" 运动, 这暴露呼吸道的病毒载体的羊水上皮流体9。这两种模式的 IUT, 静脉注射通过卵黄静脉和羊膜内, 已成为过去和正在进行的实验在我们的实验室的基础。在本议定书中, 我们详细描述了在小鼠模型中执行静脉和羊膜内 IUT 的方法。

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

在子宫移植 (IUT) 是一种独特的和多才多艺的治疗模式, 可用于引进干细胞, 病毒载体, 或任何其他物质早在妊娠期。IUT 治疗目的背后的基本原理是基于胎儿的小尺寸、胎儿的免疫不成熟、胎儿茎或祖细胞的可及性和增殖性, 以及治疗疾病或症状发作的可能性。出生前。利用胎儿的这些正常发育特性,通过IUT 提供造血干细胞 (HSC) 有可能治疗先天性血液病, 如镰状细胞疾病, 而不需要清髓性或免疫抑制的适应需要的产后 HSC 移植。同样, 在发育过程中, 多器官中祖细胞的可获得性可能允许在基因治疗或基因组编辑的病毒载体 IUT 后更有效地靶向茎/祖细胞。此外, IUT 可用于研究正常的发育过程, 包括但不限于免疫耐受的发展。小鼠模型提供了一个有价值和负担得起的手段, 以了解 IUT 的潜力和局限性之前, 临床大动物研究和最终的临床应用。在这里, 我们描述了一个协议, 以执行 IUT 在小鼠胎儿通过静脉和羊膜内路线。该协议已成功地用于阐明宫内造血干细胞移植、耐受诱导和宫内基因治疗的必要条件和机制。

生存和植入是 IUHCT 实验成功的重要措施。根据实验的特定端点, 接受 IUHCT 的胎儿可以通过 C 节或后天产前分析。平均来说, 静脉注射后的存活率从 75-100% 不等。羊膜内注射后的存活率比静脉注射的效果好, 约为 85-100%。
在我们的实验室里, 达到这些技术熟练程度的训练过程大约需要 8-12 月。为了评估获得以可重现的方式执行这?...

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

小鼠宫内移植中静脉和羊膜内植入的研究

我们描述了一个协议, 执行一个在子宫内移植 (IUT) 通过静脉注射和羊膜内注射途径的小鼠模型。该协议可用于将细胞、病毒载体和其他物质引入到独特的免疫耐受性胎儿环境中。

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 ...

该方法有助于回答胚胎学、基因治疗和干细胞移植等关键问题。这项技术的一个优点是，它能够分析不同实验疗法对穆林胎儿的影响。演示这个程序将是医生尼古拉斯安，和芭芭拉库恩斯，医生和研究员在我们的实验室。在开始手术之前，将5至10微升无菌PBS放入和从手术针头尖端中放入和退出，两到三次清除任何可能的碎屑。接下来，在适当的实验体积和浓度下用感兴趣的试剂填充针头。并在微型喷油器上三次按下模式按钮，前进到喷射校准屏幕。添加 10 或 100 毫秒的间隔以调整喷射时间，然后再按两次模式按钮。按下平衡按钮并按下脚踏板一次。然后再次踩下踏板，评估每推一次从针头中清空的溶液量。当微型喷油器校准到适当的实验体积时，将针头填充到正确的注射水平。当所有的针头都洗干净后，确认对怀孕两到六个月大的雌性小鼠的"双眼捏"缺乏反应。剃须腹部，注意不要损坏奶嘴，将动物转移到处于上部位置的加热垫上。将软膏涂抹在动物的眼睛上，用胶带将四肢固定到加热垫上。对裸露的皮肤进行消毒。然后注射局部麻醉剂，如100微升0.25%的布皮瓦卡因皮。接下来，做一到两厘米的皮肤切口，使下边界不接近一厘米的内向，并确定筋膜的中线，注意不要伤害周围的胃膜。使用Adson钳子，捏筋膜，而不抓住任何基础器官或胎儿，并打开组织用剪刀。一旦安全地进入腹腔内，将筋膜切口扩展到皮肤切口的长度，并使用棉尖施用器将肠道移动到腹部的上部，以暴露腹膜。通过切口提取子宫，仔细识别左右卵巢，以确保所有胎儿都计数。将左子宫放回腹部，以便只露出右子宫，保持非注射胎儿温暖。轻轻地抓住非占统治地位的手拇指和食指之间最横向的羊水囊。将解剖显微镜放在动物上，并调整焦点和照明，以根据需要更好地对胎儿进行可视化。对于静脉注射旋转子宫，使葡萄球菌虚荣，要注射平行于针尖。并且把针放在子宫上，以五度角刺穿子宫壁。子宫壁和羊水囊之间的尖端，将尖端直接放在葡萄线上虚荣，以几乎切线的角度，将针头滑过虚荣，直到斜面进入容器。必须保持低角，使针头不会穿透容器的后壁。当在针头上观察到血点时，踩脚踏板将感兴趣的材料的全部体积注入容器中。对于羊膜注射，旋转羊水囊，直到找到没有容器的位置。指向针垂直于子宫壁，刺穿针通过子宫蛋黄囊，羊膜囊注意不穿透任何胎儿组织。然后注入所需的适当体积的材料。在任一类型的注射后，在提供所需的体积后，从注射部位中取取针头。继续下一个胎儿，直到右子宫角的所有胎儿都注射。然后将右子宫角返回腹部。将左子宫角从腹部取出。当左角的所有胎儿都注射后，将整个子宫返回到腹部，注意避免子宫或肠道狼。使用一次性转移移液器将约两毫升无菌 PBS 分配到腹部，以替换任何不易察觉的损失。并使用4-0多糖蛋白910缝合关闭筋膜和腹部在一个连续层。然后将鼠标转移到热灯下的清洁笼中，进行监控，直到完全保持状态。在这项训练实验中，受训者接受子宫内移植的胎儿的肝脏在注射24小时后显示荧光水平较低，因为移植的GP细胞的移植率低于有经验的讲师注射的肝脏。GFP正供体细胞的流动细胞测量分析也与荧光显微镜下观察到的较低水平荧光素相关。通过羊水内部途径传递的病毒载体的能力，可传感器细胞与羊水接触。上皮细胞在妊娠日12.5胎儿携带GGFP转基因的腺病毒子宫注射48小时后转导就是例证。一旦掌握了这项技术，每只怀孕的老鼠可以在15-25分钟内完成。看完这个视频后，你应该有一个很好的理解，如何安全和有效地提供不同的治疗小鼠胎儿。

Research

JoVE Journal

Developmental Biology

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

小鼠胚胎跖骨文化：软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...