Publication Date

2019

Journal Title

Front Immunol

Abstract

Upon antigen recognition, naïve B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs), called plasma cells. Increased circulating plasma cells are reported in patients with B cell-associated malignancies, chronic graft-vs.-host disease, and autoimmune disorders. Our aim was to optimize an RNAi-based method that efficiently and reproducibly knocks-down genes of interest in human primary peripheral B cells for the targeted analysis of ASC differentiation. The unique contributions of transcriptional diversity in species-specific regulatory networks and the mechanisms of gene function need to be approached directly in human B cells with tools to hone our basic inferences from animal models to human biology. To date, methods for gene knockdown in human primary B cells, which tend to be more refractory to transfection than immortalized B cell lines, have been limited by losses in cell viability and ineffective penetrance. Our single-step siRNA nucleofector-based approach for human primary naïve B cells demonstrates reproducible knockdown efficiency (~40-60%). We focused on genes already known to play key roles in murine ASC differentiation, such as interferon regulatory factor 4 (IRF4) and AID. This study reports a validated non-viral method of siRNA delivery into human primary B cells that can be applied to study gene regulatory networks that control human ASC differentiation.

Volume Number

10

Pages

1652

Document Type

Article

Status

Faculty

Facility

School of Medicine

Primary Department

Molecular Medicine

Additional Departments

General Pediatrics

PMID

31396212

DOI

10.3389/fimmu.2019.01652


Share

COinS