BIOMAT Database Logo BIOMAT Database Logo

[Construction of CD19-CAR retroviral vector and modification of its transduction of human T-lymphocytes]. 2015

Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.

OBJECTIVE To improve the MigR1-CD19-CAR (chimeric antigen receptor) that contains a single chain variable region (scFv) which targeted to CD19 through a retroviral vector transduction efficiency of T-lymphocytes. METHODS Insert the CD19-CAR fragment into the retroviral vector (MigR1) through recombinant DNA technology, after transfecting plat-A packaging cell lines, viral supernatant was collected to transduce K562 cell line and activated human T-lymphocytes. We used flow cytometry to determine the transduction efficiency and RT-PCR to confirm the transcription of CD19-CAR gene. The ability of the transduced T cells to produce IFN-γ and TNF-α in a CD19-specific manner was measured in an enzyme-linked immunosorbent (ELISA) assay. RESULTS (1)Using MigR1-CD19-CAR retroviral vector to produce the high titer retrovirus. (2)MigR1-CD19-CAR transduction efficiency of K562 cell line was significantly higher than human T-lymphocytes (P<0.01). (3)120 min centrifugation could significantly improve transduction efficiency of T-lymphocytes to (54.5±14.6)%. (4)Transduction efficiency could be improved by deciding transduce time according to T-lymphocytes proliferation fold in vitro individually, and the highest transduction efficiency in the study was 69.3%. The CD19-CAR gene sequence was transcripted specificly with high efficiency. (5) IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased to (13 230±1 543) pg/ml and (4 217±211) pg/ml when coculture with CD19-K562 cells. CONCLUSIONS We have successfully constructed a second generation CAR which targeted to CD19 through a retroviral vector called MigR1 (MigR1-CD19-CAR). Deciding transduce time according to T-lymphocytes proliferation fold in vitro individually and 120 min centrifugation could improve the CAR transduction efficiency of T-lymphocytes. RT-PCR confirmed that the CD19-CAR gene was specificly transcripted with high efficiency. IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased when activated by target cells.

UI MeSH Term Description Entries
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012190 Retroviridae Family of RNA viruses that infects birds and mammals and encodes the enzyme reverse transcriptase. The family contains seven genera: DELTARETROVIRUS; LENTIVIRUS; RETROVIRUSES TYPE B, MAMMALIAN; ALPHARETROVIRUS; GAMMARETROVIRUS; RETROVIRUSES TYPE D; and SPUMAVIRUS. A key feature of retrovirus biology is the synthesis of a DNA copy of the genome which is integrated into cellular DNA. After integration it is sometimes not expressed but maintained in a latent state (PROVIRUSES). Leukemogenic Viruses,Leukoviruses,Oncornaviruses,Oncovirinae,Oncoviruses,Oncoviruses, Type C,RNA Tumor Viruses,Retroviruses,Type C Oncoviruses,C Oncovirus, Type,C Oncoviruses, Type,Leukemogenic Virus,Leukovirus,Oncornavirus,Oncovirus,Oncovirus, Type C,RNA Tumor Virus,Retrovirus,Tumor Virus, RNA,Tumor Viruses, RNA,Type C Oncovirus,Virus, Leukemogenic,Virus, RNA Tumor,Viruses, Leukemogenic,Viruses, RNA Tumor
D013601 T-Lymphocytes Lymphocytes responsible for cell-mediated immunity. Two types have been identified - cytotoxic (T-LYMPHOCYTES, CYTOTOXIC) and helper T-lymphocytes (T-LYMPHOCYTES, HELPER-INDUCER). They are formed when lymphocytes circulate through the THYMUS GLAND and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T Cell,T Lymphocyte,T-Cells,Thymus-Dependent Lymphocytes,Cell, T,Cells, T,Lymphocyte, T,Lymphocyte, Thymus-Dependent,Lymphocytes, T,Lymphocytes, Thymus-Dependent,T Cells,T Lymphocytes,T-Cell,T-Lymphocyte,Thymus Dependent Lymphocytes,Thymus-Dependent Lymphocyte
D014162 Transfection The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES. Transfections
D049109 Cell Proliferation All of the processes involved in increasing CELL NUMBER including CELL DIVISION. Cell Growth in Number,Cellular Proliferation,Cell Multiplication,Cell Number Growth,Growth, Cell Number,Multiplication, Cell,Number Growth, Cell,Proliferation, Cell,Proliferation, Cellular
D051603 Recoverin A neuronal calcium-sensor protein that is found in ROD PHOTORECEPTORS and CONE PHOTORECEPTORS. It interacts with G-PROTEIN-COUPLED RECEPTOR KINASE 1 in a Ca2+ dependent manner and plays an important role in PHOTOTRANSDUCTION. Cancer-Associated Retinopathy Antigen,S-Modulin,Sensitivity-Modulating Protein,Cancer Associated Retinopathy Antigen,S Modulin,Sensitivity Modulating Protein
D018941 Antigens, CD19 Differentiation antigens expressed on B-lymphocytes and B-cell precursors. They are involved in regulation of B-cell proliferation. CD19 Antigens,B Cell Antigen CD19,CD19 Antigen,Antigen, CD19
D020014 K562 Cells An ERYTHROLEUKEMIA cell line derived from a CHRONIC MYELOID LEUKEMIA patient in BLAST CRISIS. Cells, K562

Related Publications

Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration.
February 2020, The Journal of clinical investigation,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Retroviral transduction of murine primary T lymphocytes.
January 2009, Methods in molecular biology (Clifton, N.J.),
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Genetic Modification of Tumor-Infiltrating Lymphocytes via Retroviral Transduction.
January 2020, Frontiers in immunology,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Generation of CAR T-cells using γ-retroviral vector.
January 2022, Methods in cell biology,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
High-efficiency retroviral vector-mediated gene transfer into human T lymphocytes.
January 1998, Advances in experimental medicine and biology,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Immunotherapy of Hematological Malignancies of Human B-Cell Origin with CD19 CAR T Lymphocytes.
April 2024, Cells,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.
April 2022, Cancer immunology research,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
kat: a high-efficiency retroviral transduction system for primary human T lymphocytes.
January 1994, Blood,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.
April 2004, The journal of gene medicine,
Yang Wang, and Gusheng Tang, and Lili Xu, and Jie Ruan, and Hui Cheng, and Hong Zhou, and Yifei Hua, and Xiaoxia Hu, and Haihui Gu, and Baohua Qian, and Jianmin Wang, and Jianmin Yang
CD19 CAR T Cells.
December 2017, Cell,
Copied contents to your clipboard!