Abstract
Primary autologous B-lymphocytes, following ex vivo gene transfer and re-implantation, have been successfully utilized to prevent autoimmune disease and adaptive responses to therapeutic proteins in several animal models. However, efficient gene transfer to primary B cells requires use of retroviral vectors, which increase the risk of insertional mutagenesis. Here, we evaluated several alternative gene transfer approaches. Resting splenic B cells were purified and activated with LPS, and ex vivo GFP gene transfer was performed by means of nucleofection, lipofectamine, adenoviral infection, or murine retroviral infection. The Adenoviral (Ad) vectors were added to B cell cultures with or without calcium phosphate precipitation. For transfection and nucleofection, naked plasmid DNA was utilized. Nucleofection technology represents a modified electroporation technique for effective transfer of nucleic acids to the nucleus and thus enhances the efficiency of transfer particularly for primary cells. Efficiency of ex vivo gene transfer was determined by flow cytometry using GFP, CD19, and a vital dye as markers. Nucleofection yielded the highest level of gene transfer with 60-65% of B cells being GFP+. Efficiencies were 30-35% for retrovirus, 20% for Ad5/11, 15% for Ad5/35, and 5% for lipofectamine-mediated transfection. Calcium phosphate precipitation increased efficiencies for Ad vectors to 30% (Ad5/11) and 25% (Ad5/35). Lipofectamin caused the greatest cell death at 80%, followed by nucleofection (35%), and viral vector (10-15% in each case). For all methods, gene transfer efficiencies were nearly identical for B cells from C57BL/6 or C3H/HeOuJ mice. In conclusion, recent advances in gene transfer technologies provide alternatives to retroviral vectors for primary B cells. If stable gene transfer is desired, non-integrating vector systems may be combined with transposon- or phage integrase-based systems or future site-specific systems to achieve integration into the host B cell genome.
Highlights
B-lymphocytes play a crucial role in adaptive immune responses as producers of immunoglobulins and as antigen presenting cells
We describe several viral and non-viral methods for effective ex vivo gene transfer to primary murine B cells
Upon examination of green fluorescent protein (GFP) expression after ex vivo gene transfer to primary activated murine B cells, nucleofection was found to yield the highest and earliest detectable GFP signal (Figure 1). In both of the C3H/HeOuJ and C57BL/6 mice, nucleofection yielded a high efficiency of gene transfer (65–75% of B cells were GFP+), which was 3- to 5-fold higher in comparison to adenovirus serotype 5/11 (19–25%) and 5/35 (12–18%) gene transfer and 2-fold higher than retroviral (MSCV-CMV-GFP) gene transfer (24–38%) (Figure 2a,b)
Summary
B-lymphocytes play a crucial role in adaptive immune responses as producers of immunoglobulins and as antigen presenting cells. Efficient gene transfer to primary B cells has the potential to induce immune responses against tumors, thereby allowing development of novel therapeutic strategies for treatment of cancer [1,2,3,4]. We describe several viral and non-viral methods for effective ex vivo gene transfer to primary murine B cells.
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