Okitsu A, Tagawa M, Tamura Y, Kanno M, Matsubara H, Ito T, Imai K, Shigemoto K, Nakamura I, Koseki H
Department of Immunology, School of Medicine, Chiba University, Japan.
Jpn J Cancer Res. 1988 Jun;79(6):718-25. doi: 10.1111/j.1349-7006.1988.tb02228.x.
We have isolated the genomic DNA controlling the expression of murine specific melanoma antigen by employing cosmid shuttle vector and monoclonal antibody. Transfection of the cosmid library derived from mouse melanoma cells into human melanomas and repeated cell sortings of the fluorescence-bright population enabled us to enrich the antigen-positive transfectants. We rescued a 34.8 kb DNA fragment from the transfectants by in vitro packaging and showed it to be responsible for the antigen expression. However, we noticed instability of the antigen expression when the selection pressure imposed by the cell sorting was removed. This seemed to be due to the fact that the insert DNA was preferentially deleted from this cosmid vector without loss of the vector sequence itself.
我们通过使用黏粒穿梭载体和单克隆抗体，分离出了控制小鼠特异性黑色素瘤抗原表达的基因组DNA。将源自小鼠黑色素瘤细胞的黏粒文库转染至人黑色素瘤细胞中，并对荧光明亮的群体进行反复细胞分选，使我们能够富集抗原阳性转染子。我们通过体外包装从转染子中拯救出一个34.8 kb的DNA片段，并证明它负责抗原表达。然而，当去除细胞分选施加的选择压力时，我们注意到抗原表达不稳定。这似乎是由于插入DNA优先从该黏粒载体中缺失，而载体序列本身并未丢失。
