Lewis B C, Chinnasamy N, Morgan R A, Varmus H E
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Virol. 2001 Oct;75(19):9339-44. doi: 10.1128/JVI.75.19.9339-9344.2001.
We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 x 10(3) infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 10(5) IU/ml and can be concentrated by ultracentrifugation to titers of 10(7) IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the beta-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.
我们正在使用禽白血病-肉瘤病毒（ALSV）载体，在表达TVA（A亚群ALSV的受体）的转基因小鼠中建立小鼠肿瘤模型。与其他经典逆转录病毒一样，ALSV在感染宿主细胞后需要细胞分裂才能建立前病毒。相比之下，慢病毒载体能够将其病毒DNA整合到非分裂细胞的基因组中。为了在静止的TVA阳性细胞中引发肿瘤发生，我们开发了一种制备基于人免疫缺陷病毒1型（HIV-1）的慢病毒载体的系统，该载体用A亚群ALSV的包膜蛋白（EnvA）进行假型化。HIV（ALSV-A）载体保留了对靶细胞表面TVA的需求，并且可以以5×10³感染单位（IU）/毫升的滴度产生。通过插入来自HIV-1 pol基因的中央多聚嘌呤序列（cPPT）并去除EnvA的细胞质尾巴，可以产生滴度接近10⁵IU/毫升的假型，并且可以通过超速离心浓缩至10⁷IU/毫升的滴度。HIV（ALSV-A）还能感染来自转基因小鼠的胚胎成纤维细胞，其中TVA的表达由β-肌动蛋白启动子驱动。此外，当这些成纤维细胞对ALSV载体感染具有抗性时，这种慢病毒假型在细胞周期停滞后能有效地感染它们。该系统可能有助于将基因导入成年TVA转基因动物的体细胞中，并允许在体内评估分化细胞类型中基因表达改变的影响。
