Jung Daniel, Néron Sonia, Drouin Mathieu, Jacques Annie
Héma-Québec R and D, Québec, Canada.
J Immunol Methods. 2005 Sep;304(1-2):78-87. doi: 10.1016/j.jim.2005.06.010.
The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34+ progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40-CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than naïve B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.
无法有效地将基因导入正常细胞（如人类B淋巴细胞）限制了对其在细胞生长、分化和存活方面功能的表征。最近的研究表明，一种新的腺病毒载体Ad5/F35能够有效地转导人类造血CD34+祖细胞。在本研究中，我们比较了Ad5/F35载体与亲本载体Ad5在人类B淋巴细胞中的基因转移效率。从健康个体获得的外周血B细胞使用CD40-CD154系统进行体外培养。正常B淋巴细胞用均含有绿色荧光蛋白（GFP）报告基因的复制缺陷型Ad5和Ad5/F35进行感染，通过流式细胞术监测转导效率。Ad5效率极低，仅感染约5%的人类B淋巴细胞。相比之下，Ad5/F35可转导高达60%的人类B淋巴细胞，并且在感染后长达5天可检测到GFP表达。重要的是，Ad5/F35转导后，B淋巴细胞的增殖、活力和抗体分泌等生理功能未受影响。最后，我们观察到记忆B淋巴细胞比初始B淋巴细胞更易受到Ad5/F35感染。因此，我们的结果表明腺病毒载体Ad5/F35是用于B淋巴细胞生成中基因功能表征的有效工具。
