Establishment of the CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse
10.3760/cma.j.issn.1009-9921.2015.12.003
- VernacularTitle:CD19基因过表达K562细胞株及NOD-SCID小鼠皮下移植瘤模型的建立
- Author:
Yang WANG
;
Hui CHENG
;
Keting CHEN
;
Miaoxia HE
;
Jie RUAN
;
Gusheng TANG
;
Jianmin YANG
- Publication Type:Journal Article
- Keywords:
Gene,CD19;
Transfection;
K562 cell;
Mice,NOD-SCID;
Xenograft model
- From:
Journal of Leukemia & Lymphoma
2015;24(12):715-721
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a MigR1-CD19 recombinant vector which contains CD19 gene, and to establish a CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse.Methods The CD19 gene was inserted into the retroviral vector (MigR1) through recombinant DNA technology after transfection into Plat-A packaging cells, and viral supernatant was collected to transduce K562 cell line repeatedly to obtain stable transduction CD19-K562 cell line.Flow cytometry was used to determine the transduction efficiency and RT-PCR was used to confirmed CD19 gene expression.Cell proliferation and apoptosis were detected by cell count and Annexin V/PI, respectively.Then the subcutaneous xenograft subtype of CD19-K562-a cell line was constructed through subcutaneous inoculation and was cultured in vitro and in vivo.Then its subcutaneous xenograft model in NOD-SCID mouse was established.The characteristics of CD19-K562-a cells were detected by RT-PCR, Wright staining and immunohistochemistry.Results MigR1-CD19 recombinant vector was successfully constructed, and the CD19 positive efficiency of K562 cell line was (99.80±0.17) ％ through retrovirus centrifugation transduction.The transduction and passage had no effects on proliferation and apoptosis of CD19-K562 cells.The CD19-K562-a cell line was constructed after CD19-K562 cells were injected subcutaneously and were passaged in vitro and in vivo.The CD19 positive efficiency of the xenograft subtype CD19-K562-a cell line was (99.78± 0.04) ％.CD19-K562-a and CD19-K562 cells were in an undifferentiated state.NOD-SCID subcutaneous xenografts were established through subcutaneous inoculation of CD19-K562-a cells.CD19 in the CD19-K562-a subcutaneous xenografts was positive, while it was negative in its counterparts K562 cells.Conclusion The CD19-K562 cell line over-expressing CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse are successfully established.
