This CORE will provide expertise in the preparation of both MuLV retroviral and lentiviral vectors and in the use of these vectors and in the use of these vectors in cell culture and animal model experimentation. MuLV vector preparations will be derived from stable producer cell clones or from 293T cells which have been transiently transfected with vector and packaging (helper plasmids). Titers of the conditioned medium from producer cell clones or 293T cells for vectors containing the GFP marker will be determined by FACS analysis of the transduced population of cells; e.g., ET3 or HeLa cells. In the context of these analysis, DNA will be harvested from cells which are 100% transduced and analyzed to evaluated transmission of the pro-viral vector genome using the packaging signal as a probe since these sequences will be present in all transmissible vector particles. Titers of vectors lacking the marker gene will be determined by Southern blot analysis of DNA from target cells transduced with various dilutions of the vector preparations. Lentiviral vectors are produced by co-transfection of 293T cells with a vector, GAG/POL helper, and ENV helper plasmids. Particles pseudotyped with the VSV-G envelope protein will be concentrated by ultra-centrifugation. Tittering is determined by transmission of the GFP marker of the intact pro-viral genome is determined by Southern blot analysis as described above. MuLV retroviral vector preparations are screened for replication competent retrovirus (RCR) by marker rescue from Mus. dunni cells. Lentiviral vector preparations are screened for RCR by measuring p24, a protein encoded by the GAG region, in exponentially growing lymphoid cells that are sensitive to infection by replication competent HIV. This CORE is responsible for maintaining supplies of cell lines, including retroviral producer clones, packaging cell lines as well as other lines that are required for the analysis of the vector preparations. The CORE will also be responsible for maintaining a computerized database for over 800 plasmids and for a variety of cell lines.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL053749-07
Application #
6346221
Study Section
Project Start
2000-09-01
Project End
2001-08-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
7
Fiscal Year
2000
Total Cost
$202,050
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Zhao, Hui Fen; Abraham, Allistair; Kim, Yoon-Sang et al. (2017) Lentiviral Transfer of ?-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine ?-Thalassemia. Mol Ther 25:593-605
De Ravin, Suk See; Wu, Xiaolin; Moir, Susan et al. (2016) Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8:335ra57
Abraham, Allistair; Kim, Yoon-Sang; Zhao, Huifen et al. (2016) Increased Engraftment of Human Short Term Repopulating Hematopoietic Cells in NOD/SCID/IL2r?null Mice by Lentiviral Expression of NUP98-HOXA10HD. PLoS One 11:e0147059
Wielgosz, Matthew M; Kim, Yoon-Sang; Carney, Gael G et al. (2015) Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy. Mol Ther Methods Clin Dev 2:14063
Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen et al. (2015) Amelioration of murine sickle cell disease by nonablative conditioning and ?-globin gene-corrected bone marrow cells. Mol Ther Methods Clin Dev 2:15045
Zhou, Sheng; Bonner, Melissa A; Wang, Yong-Dong et al. (2015) Quantitative shearing linear amplification polymerase chain reaction: an improved method for quantifying lentiviral vector insertion sites in transplanted hematopoietic cell systems. Hum Gene Ther Methods 26:4-12
Urbinati, Fabrizia; Hargrove, Phillip W; Geiger, Sabine et al. (2015) Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells. Exp Hematol 43:346-351
Treanor, Louise M; Zhou, Sheng; Janke, Laura et al. (2014) Interleukin-7 receptor mutants initiate early T cell precursor leukemia in murine thymocyte progenitors with multipotent potential. J Exp Med 211:701-13
Griffith, Linda M; Cowan, Morton J; Notarangelo, Luigi D et al. (2014) Primary Immune Deficiency Treatment Consortium (PIDTC) report. J Allergy Clin Immunol 133:335-47
De Ravin, Suk See; Gray, John T; Throm, Robert E et al. (2014) False-positive HIV PCR test following ex vivo lentiviral gene transfer treatment of X-linked severe combined immunodeficiency vector. Mol Ther 22:244-245

Showing the most recent 10 out of 152 publications