Name: using the blt humanized mouse as a stem cell based gene therapy tumor model
Uploaded: 2012-12-18T12:00:00
Description: Watch this Scientific Journal Video about Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model at JoVE.com

Мы хотели бы поблагодарить Элвин и Ларри Уэлч Pang за техническую помощь. Эта работа была частично финансируется Национальным институтом здоровья (NIH) награды P50 CA086306, Калифорнийского института регенеративной медицины (CIRM) гранты RC1-00149-1 и RS1-00203-1; CIRM Новый факультет премии RN2-00902-1 , Caltech-UCLA Объединенный центр Поступательное медицины, UCLA Центра исследований СПИДа (CFAR) NIH / NIAID AI028697, UCLA AIDS Institute, CIRM инструментов и технологий RT1-01126, и Научно-исследовательский UC Multicampus Программы и инициативы из Калифорнийского Центр противовирусных лекарственных препаратов (число MRPI-143226).

А. Генерация BLT мышей Поколения BLT мышей разделили на три (3) части: (1) обработка ткани плода и подготовке CD34 человека гемопоэтических стволовых клеток, (2) трансплантации человеческих тканей, и (3) вторичные пересадки. Для всех протоколов, мы представили таблицу 1 с ре...

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	<li>McCune, J. M., Namikawa, R., Kaneshima, H., Shultz, L. D., Lieberman, M., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCID-hu+mouse:+murine+model+for+the+analysis+of+human+hematolymphoid+differentiation+and+function.">The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function.</a> Science. 241 (4873), 1632-1639 (1988).</li><li>Bristol, G. C., Gao, L. Y., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+maintenance+of+SCID-hu+mice+for+HIV+research.">Preparation and maintenance of SCID-hu mice for HIV research.</a> Methods. 12 (4), 343-347 (1997).</li><li>Mosier, D. E., Gulizia, R. J., Baird, S. M., Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+a+functional+human+immune+system+to+mice+with+severe+combined+immunodeficiency.">Transfer of a functional human immune system to mice with severe combined immunodeficiency.</a> Nature. 335 (6187), 256-259 (1988).</li><li>Melkus, M. W., Estes, J. D., Padgett-Thomas, A., Gatlin, J., Denton, P. W., Othieno, F. A., Wege, A. K., Haase, A. T., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+mount+specific+adaptive+and+innate+immune+responses+to+EBV+and+TSST-1.">Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.</a> Nat. Med. 12 (11), 1316-1322 (2006).</li><li>Denton, P. W., Estes, J. D., Sun, Z., Othieno, F. A., Wei, B. L., Wege, A. K., Powell, D. A., Payne, D., Haase, A. T., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiretroviral+pre-exposure+prophylaxis+prevents+vaginal+transmission+of+HIV-1+in+humanized+BLT+mice.">Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.</a> PLoS Med. 5 (1), e16 (2008).</li><li>Sun, Z., Denton, P. W., Estes, J. D., Othieno, F. A., Wei, B. L., Wege, A. K., Melkus, M. W., Padgett-Thomas, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarectal+transmission,+systemic+infection,+and+CD4++T+cell+depletion+in+humanized+mice+infected+with+HIV-1.">Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.</a> J. Exp. Med. 204 (4), 705-714 (2007).</li><li>Shimizu, S., Hong, P., Arumugam, B., Pokomo, L., Boyer, J., Koizumi, N., Kittipongdaja, P., Chen, A., Bristol, G., Galic, Z., Zack, J. A., Yang, O., Chen, I. S., Lee, B., An, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+short+hairpin+RNA+potently+down-regulates+CCR5+expression+in+systemic+lymphoid+organs+in+the+hu-BLT+mouse+model.">A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.</a> Blood. 115 (8), 1534-1544 (2010).</li><li>V, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+HIV+Latency+in+Humanized+BLT+Mice.">Generation of HIV Latency in Humanized BLT Mice.</a> J. Virol. 86 (1), 630-634 (2012).</li><li>Vatakis, D. N., Koya, R. C., Nixon, C. C., Wei, L., Kim, S. G., Avancena, P., Bristol, G., Baltimore, D., Kohn, D. B., Ribas, A., Radu, C. G., Galic, Z., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+from+antigen-specific+CD8+T+cells+generated+in+vivo+from+genetically+engineered+human+hematopoietic+stem+cells.">Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (51), 1408-1416 (2011).</li></ol>

Изменение BLT гуманизированные модель мыши в сочетании с в естественных условиях ПЭТ являются мощным инструментом для изучения хронических заболеваний человека. Эта система занимает BLT модели мыши и продвигает ее пределами ограниченные возможности для исследований ВИЧ-инфекции. ...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии (по желанию) </td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 10010049 </td><td></td></tr><tr><td> RPMI 1640 </td><td></td><td> A1049101 </td><td></td></tr><tr><td> Искова </td><td></td><td> 12440061 </td><td></td></tr><tr><td> Эмбриональной телячьей сыворотки </td><td></td><td> 16000085 </td><td></td></tr><tr><td> Коллагеназы типа IV </td><td> Sigma Aldrich </td><td> C5138 </td><td></td></tr><tr><td> Гиалуронидаза </td><td></td><td> H3506 </td><td></td></tr><tr><td> ДНКазы </td><td> Roche Diagnostics </td><td> 10104159001 &lt;/ TD&gt; <td></td></tr><tr><td> Piptazo (Zosyn) </td><td> Pfizer </td><td></td><td> <a href="http://labeling.pfizer.com/showlabeling.aspx?id=416" target="_blank">Пиперациллин / тазобактам комбинации</a> </td></tr><tr><td> Ficoll (Ficoll-Пак Плюс) </td><td> GE Healthcare Life Sciences </td><td> 17-1440-02 </td><td></td></tr><tr><td> MACS буфера </td><td> Miltenyi Biotech </td><td> См. комментарии </td><td> MACS буфера: PBS с добавлением 0,5% эмбриональной телячьей сыворотки, 2 мМ цитрата декстрозы Формула- </td></tr><tr><td> CD34 Microbead Kit </td><td></td><td> 130-046-702 </td><td></td></tr><tr><td> LS Столбцы </td><td></td><td> 130-042-401 </td><td></td></tr><tr><td> Retronectin </td><td> Takara </td><td> T100A </td><td></td></tr><tr><td&gt; Matrigel </td><td> BD Biosciences </td><td> 354263 </td><td></td></tr><tr><td> Isofluorane </td><td> Baxter </td><td></td><td> <a href="http://www.baxter.com/healthcare_professionals/products/forane.html" target="_blank">http://www.baxter.com/healthcare_professionals/products/forane.html</a> </td></tr><tr><td> Carprofen (Римадил) </td><td> Pfizer Animal Health </td><td></td><td> <a href="https://www.rimadyl.com/default.aspx" target="_blank">https://www.rimadyl.com/default.aspx</a> </td></tr></tbody></table>

using the blt humanized mouse as a stem cell based gene therapy tumor model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

Блок-схема процесса трансплантации показано на рисунке 1а. Картину твоих / жилая имплантата показано на рисунке 1b. Тимуса ткани развивается нормально и имеет физиологическое распределение человеческих CD4 и CD8 Т-клеток. После восстановления, животные несут иммунной си?...

Watch this Scientific Journal Video about Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model at JoVE.com

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Поколения и характеристику опухоли специфические Т-клетки использованием гуманизированных мышей описано здесь. Человека тимуса ткани и генетически измененных человеческих гемопоэтических стволовых клеток пересаживают в ослабленным иммунитетом мышей. В результате восстановление инженерных человеческой иммунной системы, позволяющей в естественных экспертизы противоопухолевого иммунного ответа.

Использование BLT Humanized мышь в качестве стволовых клеток на основе модели генной терапии опухолей

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Research

JoVE Journal

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Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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