Construction of CD19-CAR retroviral vector and modification of its transduction of human T-lymphocytes.
- VernacularTitle:利用MigR1质粒构建CD19-CAR逆转录病毒载体优化其对人原代T细胞的转染效率
- Author:
Yang WANG
1
;
Gusheng TANG
1
;
Lili XU
1
;
Jie RUAN
1
;
Hui CHENG
1
;
Hong ZHOU
1
;
Yifei HUA
1
;
Xiaoxia HU
1
;
Haihui GU
1
;
Baohua QIAN
1
;
Jianmin WANG
1
;
Jianmin YANG
1
Author Information
- Publication Type:Journal Article
- MeSH: Antigens, CD19; Cell Proliferation; Flow Cytometry; Genetic Vectors; Humans; K562 Cells; Recoverin; Retroviridae; T-Lymphocytes; Transfection
- From: Chinese Journal of Hematology 2015;36(4):331-336
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSInsert 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.
CONCLUSIONWe 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.
