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

We would like to thank Alvin Welch and Larry Pang for their technical assistance. This work was funded in part by the National Institutes of Health (NIH) award P50 CA086306, the California Institute for Regenerative Medicine (CIRM) grants RC1-00149-1 and RS1-00203-1; CIRM New Faculty Award RN2-00902-1, the Caltech-UCLA Joint Center for Translational Medicine, UCLA Center for AIDS Research (CFAR) NIH/NIAID AI028697, the UCLA AIDS Institute, the CIRM Tools and Technology Award RT1-01126, and the UC Multicampus Research Program and Initiatives from the California Center for Antiviral Drug Discovery (number MRPI-143226).

A. Generation of BLT mice
The generation of BLT mice is divided into three (3) parts: (1) processing of fetal tissue and preparation of CD34 human hematopoietic stem cells, (2) transplantation of human tissue, and (3) secondary transplant. For all the protocols, we have provided Table 1 with reagent information.
1. Tissue Processing and Isolation of CD34 Human Hematopoietic Stem Cells
Fresh fetal tissue or tissue shipped overn...

<ol>
	<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>

The modified BLT humanized mouse model coupled with in vivo PET imaging are powerful tools to study chronic human diseases. This system takes the BLT mouse model and advances it beyond the limited scope for HIV research. In addition, it is a great system in which we can examine various gene therapy protocols as well as diagnostic techniques before they can reach the clinical setting. The latter coupled with the low cost of using mice versus primates makes this a very useful model.
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<table border="1">
 <tbody>
 <tr>
 <td>Name 				of the reagent</td>
 <td>Company</td>
 <td>Catalogue 				number</td>
 <td>Comments 				(optional)</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>Iscove's</td>
 <td></td>
 <td>12440061</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal 				Calf Serum</td>
 <td></td>
 <td>16000085</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase 				type IV</td>
 <td>Sigma 				Aldrich</td>
 <td>C5138</td>
 <td></td>
 </tr>
 <tr>
 <td>Hyaluronidase</td>
 <td></td>
 <td>H3506</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse 				I</td>
 <td>Roche 				Diagnostics</td>
 <td>10104159001</td>
 <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"> Piperacillin/tazobactam 				combination </a></td>
 </tr>
 <tr>
 <td>Ficoll 				(Ficoll-Paque Plus)</td>
 <td>Ge 				Healthcare Life Sciences</td>
 <td>17-1440-02</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS 				Buffer</td>
 <td>Miltenyi 				Biotech</td>
 <td>See 				comments</td>
 <td>MACS 				buffer: PBS supplemented with 0.5% fetal bovine serum and 2 mM 				citrate dextrose formula-A </td>
 </tr>
 <tr>
 <td>CD34 				Microbead Kit</td>
 <td></td>
 <td>130-046-702</td>
 <td></td>
 </tr>
 <tr>
 <td>LS 				Columns</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>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 				(Rimadyl)</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>).

A flow chart of the transplantation process is shown in Figure 1A. A picture of the thy/liv implant is shown in Figure 1B. The thymic tissue develops normally and has a physiological distribution of human CD4 and CD8 T cells. Following reconstitution, the animals carry a human immune system with normal distribution of CD4, CD8 T cells and other immune cell lineages.
The discrepancy between tumor size and live tissue is shown in Figure 2. ...

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

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

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

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