Moloney leukemia virus-based vectors can be generated in cells that express the products of three retroviral genes, gag, pol, and env. There are a number of cell lines such as PG13 and FLYA13 (2), known as packaging cells, that have been established that stably express these genes. When these cells are transfected with vector DNA, they will generate retroviral transducing particles in the supernatant of the cells. The transducing particles can be produced by transient transfection of the packaging cells or from cells that have stably integrated the vector DNA into the packaging cell line. This is particularly useful if the vector will be required in large amounts for an extended period of time. The titer that can be achieved from transient transfection of DNA will be proportional to the transfection efficiency. In this respect, cells derived from the human embryonic kidney cell line 293 (3) are particularly useful because they can be transfected very efficiently (typically 90-99%) (4-7). Here I outline a method to generate vector transiently. Methods to generate stable packaging cell lines can be found elsewhere (8).
|Original language||English (US)|
|Number of pages||8|
|Journal||Methods in molecular biology (Clifton, N.J.)|
|State||Published - 2004|