Bauer T R, Hickstein D D
Medical Research Service, VA Puget Sound Health Care System, Seattle, WA 98108, USA.
Hum Gene Ther. 1997 Feb 10;8(3):243-52. doi: 10.1089/hum.1997.8.3-243.
To investigate conditions for improving transduction efficiencies of human hematopoietic stem or progenitor cells using retroviral vectors, we constructed a retroviral vector containing a modified murine CD4 cDNA reporter gene with a truncated cytoplasmic domain to prevent signaling. The advantages of using this truncated murine CD4 reporter gene include: (i) CD4 is well characterized with well-known cell signaling pathways, (ii) truncation of the cytoplasmic domain of CD4 has been demonstrated to abrogate signaling, (iii) the truncated murine CD4 is easily detectable on the cell surface with no cross-reaction to human CD4, (iv) a variety of monoclonal antibodies directed against the murine CD4 molecule are available commercially, and (v) expression of a truncated CD4 molecule in a transgenic mouse in vivo does not interfere with hematopoiesis. We cloned the truncated murine CD4 reporter gene into the retroviral vector LXSN, packaged this vector using PG13 retrovirus packaging cells, and transduced hematopoietic cell lines representing erythroid, myeloid, megakaryocyte, and lymphoid lineages using vector-containing medium harvested from the murine CD4 producer line. After seven daily exposures to vector-containing medium, all cell lines expressed murine CD4 on the cell surface, and 5-7% of human CD34+ cells expressed murine CD4 on the cell surface after 3 days of exposure to murine CD4 vector-containing medium. Colony-forming cell assays assessing progenitor cells demonstrated the presence of transduced cells in the CD34+ population. These results demonstrate the utility of using a modified murine CD4 gene in a retroviral vector to allow optimization of in vitro transduction conditions of human hematopoietic cells and to facilitate identification of the lineages that have been transduced using different growth factors, prior to clinical trials using retroviral vectors.
为了研究使用逆转录病毒载体提高人类造血干细胞或祖细胞转导效率的条件，我们构建了一种逆转录病毒载体，其包含一个修饰的鼠类CD4 cDNA报告基因，该基因具有截短的胞质结构域以防止信号传导。使用这种截短的鼠类CD4报告基因的优点包括：（i）CD4具有特征明确的知名细胞信号通路；（ii）已证明截短CD4的胞质结构域可消除信号传导；（iii）截短的鼠类CD4在细胞表面易于检测，且与人CD4无交叉反应；（iv）有多种针对鼠类CD4分子的单克隆抗体可商购；（v）在体内转基因小鼠中截短的CD4分子的表达不干扰造血作用。我们将截短的鼠类CD4报告基因克隆到逆转录病毒载体LXSN中，使用PG13逆转录病毒包装细胞包装该载体，并使用从鼠类CD4产生细胞系收获的含载体培养基转导代表红系、髓系、巨核细胞系和淋巴系的造血细胞系。在每天暴露于含载体培养基7天后，所有细胞系在细胞表面均表达鼠类CD4，并且在暴露于含鼠类CD4载体培养基3天后，5 - 7%的人类CD34+细胞在细胞表面表达鼠类CD4。评估祖细胞的集落形成细胞测定表明在CD34+群体中存在转导细胞。这些结果证明了在逆转录病毒载体中使用修饰的鼠类CD4基因的实用性，以优化人类造血细胞的体外转导条件，并在使用逆转录病毒载体进行临床试验之前，便于鉴定使用不同生长因子转导的谱系。
