Abstract
B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9(-/-) B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9(-/-) mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9(-/-) B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.
Highlights
In germinal center B cells, some DNA repair factors involved in mismatch repair (MMR) or base excision repair (BER) are diverted from their normal roles in preserving genomic integrity to increasing DNA sequence diversity within the Ig locus [4]
The mice containing loxP targeted to Rad9 were crossed with mice in which Cre transcription is under the control of the CD19 promoter [28]
We have documented that Rad9 is required for B cell proliferation (Fig. 3), Ig generation, and class switch recombination (CSR) (Fig. 4)
Summary
Generation of Rad9Tar/TarCD19cre/ϩ Mice—B cell-specific and Rad9-deficient mice were generated by crossing Rad9Tar/Tar 129SvEv strain mice [15] with CD19-Cre knock-in C.129P2-Cd19tm1(cre)Cgn/J strain mice expressing Cre under control of the endogenous CD19 promoter (The Jackson Laboratory, Bar Harbor, ME). Flow Cytometric Analysis—Lymphoid cells stained with antiB220-PE, anti-CD19-FITC, anti-B220-FITC, anti-CD23-FITC, anti-CD21-PE, anti-IgD-PE, anti-CD43-PE, anti-IgM-APC (Allophycocyanin) (Pharmingen), and anti-IgG-CyTM5 (Jackson ImmunoResearch Laboratories, West Grove, PA) were analyzed by FACSCalibur cytometer (BD Biosciences). BrdU Uptake Assays—Purified splenic B cells (106 cells/ml) were cultured in medium with IL-4 and LPS for 2 days. Cells were processed and probed with FITC-conjugated anti-BrdU antibody (BD Biosciences) and stained with propidium iodide (PI). Cells were stained with annexin V-FITC (Jingmei Biotech, Shenzhen, Guangdong, China) and PI for 15 min at room temperature and subjected to flow cytometric analysis. The cells were harvested and stained with Cy5-labeled monoclonal rat anti-mouse IgG and analyzed using a FACSCalibur cytometer. Statistical Analysis—The significance of the difference was calculated by Student’s t test, and p Ͻ 0.05 was considered statistically significant
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
