Su Y A, Ray M E, Lin T, Seidel N E, Bodine D M, Meltzer P S, Trent J M
Laboratory of Cancer Genetics, National Center for Human Genome Research, NIH, Bethesda, Maryland 20892-4470, USA.
Cancer Res. 1996 Jul 15;56(14):3186-91.
We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.
我们已经开发出一种通过逆转录病毒转导来逆转恶性表型的单染色体抑制的通用策略。我们的方法包括将携带逆转录病毒表达载体的cDNA文库导入6号染色体抑制的黑色素瘤亚系UACC - 903(+6) [J. M. 特伦特等人，《科学》（华盛顿特区），247: 568 - 571, 1990]。该cDNA文库由从受抑制的UACC - 903(+6)细胞中分离的聚腺苷酸化RNA构建而成，包装成高滴度的双嗜性逆转录病毒颗粒，并转导到UACC - 903(+6)细胞中。通过在软琼脂中分离集落形成细胞来选择回复组氨酸(R)转导子。从软琼脂培养物中总共挑选了121个大（>150微米）的菌落，其中121个中的18个（15%）被确立为永久亚系。回复亚系在琼脂平板上接种时显示出7 - 58%的克隆效率，相比之下，UACC - 903(+6)亚系的克隆效率<0.05%。所有18个回复亚系，称为SRS1 - SRS18（“用于抑制的回复子选择”），显示出群体倍增时间缩短，18个中有9个在单层中形成集落，类似于亲代（未受抑制）细胞系。对于一个回复亚系，已经完成了将细胞注射到无胸腺裸鼠中以逆转受抑制表型的初步证据。Southern分析表明逆转录病毒载体序列在所有18个亚系中均有整合。这种方法应有助于鉴定参与恶性黑色素瘤致瘤表型的基因，并且很容易适用于其他模型系统。
