Soft agar cloning

Oberly, T.J., Rexroat, M.A., Bewsey, B.J., Richardson, K.K. Michaelis, K.C. (1990) An evaluation of the CHO/HGPRT mutation assay involving suspension cultures and soft agar cloning results for 33 chemicals. Environ, mol. Mutag., 16, 260-271... 

Hamburger, A. W., Jones, S. E. and Salmon, S. E. (1980). Soft-agar cloning of cells from patients with lymphoma. Prog. Clin. Biol. Res. 48,43-52. 

Initially, we became attracted to the potential of L5178Y. cells because of a number of characteristics which would seem to favor their use in an in vitro mammalian cell mutational assay system. These include growth in suspension culture, a short generation time of 11 hr, and a cloning efficiency of up to 100% in a simple soft-agar cloning medium. 

These methods are, however, largely unnecessary when it is desired to clone continuous cell lines. Thus a dilute suspension of HeLa cells (containing about 100 cells in 5 ml) will grow up to form colonies, each derived from a single cell. It is important to prevent the movement of these cells (and hence mixing of clones) and sometimes they are overlayered with soft agar. 

The above mentioned cell lines can be cloned under appropriate conditions in soft agar. Upon addition of high concentrations of a potent glucocorticoid, for example, dexamethasone at 0.1-1 /rM concentrations the vast majority of S49.1 or CEM-7 cells die but a few cells form colonies. These clones are completely resistant to the cytolytic hormone effect. Resistance is a stable heritable trait which arises randomly in the cell population treatment with mutagens increases the rate at which resistant cells come up [20,53]. 

Cloning by the soft agar method is carried out in petri dishes 3 cm in diameter that contain a layer of normal spleen feeder cells (10 per plate) in 5% agar, over which is then layered a dilution of the hybrid cells (obtained from positive wells) suspended in a medium containing 20% fetal calf serum in 2.5% agar. A range of different dilutions of the hybrid... 

Similar cloning of normal cultures was unsuccessful, and none of the normal mass cultures derived from hamster embryos, and tested for colony formation in soft agar or formation of tumors in "nude" mice were positive. 

Baby Syrian hamster kidney BHK 21, Clone 13 Growth in soft agar 123 66-69... 

In our experience, any Drosophila cell line can be cloned by limiting dilution (in 96-well plates) provided the growth of isolated cells is supported by the presence of a suspension of X-rayed feeder cells. Some fines (notably S2) can be cloned in soft agar, at a considerable saving of labor and money. Detailed procedures for both techniques have been published recently (Cherbas et al. 1994 Cherbas and Cherbas 1998). 

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