B-hCD19-luc GFP A20

Basic Information

Description

The mouse Cd19 gene was replaced by human CD19 coding sequence in B-hCD19-luc GFP A20 cells. Human CD19 is highly expressed on the surface this cell. And this cell expresses firefly luciferase as a marker.

Targeting strategy

Gene targeting strategy for B-hCD19-luc GFP A20 cells. The exogenous promoter, human CD19 coding sequence and luciferase was inserted to replace part of murine Cd19 gene in B-hCD19-luc GFP A20 cells. Mouse Cd19 transcript was disrupted, the protein no longer be expressed.

Protein expression analysis

CD19 expression analysis in B-hCD19-luc-GFP A20 cells by flow cytometry. Single cell suspensions from B-hCD19-luc-GFP A20 were stained with anti-CD19 antibody (anti-hCD19 antibody BioLegend, Cat.302212; anti-mCD19 antibody, BioLegend, Cat.15555). Human CD19 was detected on the surface of B-hCD19-luc-GFP A20 cells but not wild-type A20 cells. And mouse CD19 was not detected on B-hCD19-luc-GFP A20 cells.

In vitro luciferase activity of B-hCD19-luc-GFP A20 cells

Luminescence signal intensity of B-hCD19-luc-GFP A20 cells. Cell lysates of wild-type A20 and B-hCD19-luc-GFP A20 cells were measured using the Bright-GloTM luciferase Assay (Promega, Catalog No. E4030). B-hCD19-luc-GFP A20 cells have a strong luminescence signal that is not present in wild-type A20 cells.

Quantitative imaging data & body weight changes

Growth kinetics of B-hCD19-luc-GFP A20 tumors determined by bioluminescence imaging (BLI). B-hCD19-luc-GFP A20 cells were injected into the tail vein of wild-type BALB/c mice (9-week-old). Signal intensity and body weight were measured twice a week. (A) Signal intensity. (B) Body weight. (C) Raw bioluminescence images. These results indicate that B-hCD19-luc-GFP A20 cells can be used for in vivo efficacy evaluation. Values are expressed as mean ± SEM.

Growth kinetics of B-hCD19-luc-GFP A20 tumors determined by bioluminescence imaging (BLI). B-hCD19-luc-GFP A20 cells were injected into the tail vein of wild-type BALB/c mice (9-week-old). Signal intensity and body weight were measured twice a week. (A) Signal intensity. (B) Body weight. (C) Raw bioluminescence images. These results indicate that B-hCD19-luc-GFP A20 cells can be used for in vivo efficacy evaluation. Values are expressed as mean ± SEM.

Tumor growth curve & Body weight changes

Subcutaneous homograft tumor growth of B-hCD19-luc-GFP A20 cells. B-hCD19-luc-GFP A20 cells and wild-type A20 cells were subcutaneously implanted into BALB/c mice (7-9 weeks old). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². As shown in panel A, B-hCD19-luc-GFP A20 cells were able to establish tumors in vivo and can be used for efficacy studies. The consistency of the #1-E04 tumor is good, but the drug administration window is short.

Protein expression analysis of tumor cells

B-hCD19-luc-GFP A20 cells (5×10⁵) and wild-type A20 cells (5×10⁵) were subcutaneously implanted into BALB/c mice (female, 7-9 weeks old, n=8), and on 24 days post inoculation, tumor cells were harvested and assessed for human CD19 expression by flow cytometry. As shown, human CD19 was highly expressed on the surface of tumor cells. Therefore, B-hCD19-luc-GFP A20 cells can be used for in vivo efficacy studies of novel CD19 therapeutics.

Quantitative imaging data & body weight changes

Growth kinetics of B-hCD19-luc-GFP A20 tumors determined by bioluminescence imaging (BLI). B-hCD19-luc-GFP A20 cells and wild-type A20 cells were subcutaneously implanted into BALB/c mice (7-9 weeks old). Signal intensity and body weight were measured twice a week. (A) Signal intensity. (B) Raw bioluminescence images. These results indicate that B-hCD19-luc-GFP A20 cells can be used for in vivo efficacy evaluation. Values are expressed as mean ± SEM.