Transfectant colonies

Fig. 4.2 Productivity distribution of antibody concentrations for primary GS-CHO transfectants. Ninety-two primary GS-CHO transfectant colonies were transferred from 96-well to 24-well plates and grown for 14 days the mean concentration at harvest was 48 mg L A log-normal probability density function was fitted to the antibody concentration data.

An assessment of the transfection efficiency of human skin fibroblasts, compared to transfection of hamster lung fibroblasts and Human HeLa cells, indicated that the protocols we used are not suitable to obtain stable transfected skin fibroblasts. We concluded this from the fact that no stable transfected colonies of human skin fibroblasts were obtained, regardless of being CPTl-defective or being from a patient with a disorder unrelated to P-oxidation. Only transient expression with the Lipofectamine method was successful. We found an efficiency of 0.0-0.2 % of the cells which survived transfection without selection. However, compared to the efficiencies of transfection in hamster lung fibroblasts (5-10% in a parallel experiment), this still was low. Therefore, for long-term apphcations it is clear that different procedures should be used than the ones we applied here. 

The isolation of transfectant colonies can be carried out in about 2—3 weeks, when transformant colonies of medium size have formed (such as the ones seen in Fig. 7). These colonies can be observed under the microscope, but they should also be visible to the naked eye when looking fi-om below into the flask. 

Figure 26.4 Typical colony of transformed airway epithelial cells. The formation of a high density colony growing in a monolayer of non-transformed cells 1 week after transfection ([21], Figure 26.1A). After several weeks, the non-transformed cells die and come off the dish.

Hogge, G.S., Burkholder, J., Culp, J., Dubielzig, R.R., Albertini, M.R., Keller, E.T. et al. (1998) Development of human granulocyte macrophage-colony stimulating factor transfected tumor cell vaccines for the treatment of spontaneous canine cancer. Hum. Gene Ther., 9, 1851-1861. 

Number of drug resistant colonies formed following culture of cells obtained from collagenase-dissociated organs with metastatic foci composed of tumor cells transfected with a selective vector. 

In general, cells transfected with DNA that contains transforming genes, derived from oncogenic viruses, lose the growth feature called contact inhibition, which limits their growth and proliferation. These transfectants can be visualized under the microscope and isolated as colonies. 

The power of the screening or selection method ultimately delineates the extent to which the sequence space can be explored. Screening usually involves the physical separation of mutants identified on some phenotypic change such as colony size or color. It is essentially a brute force approach that is amenable to amplification by robotics and is limited to identification of mutants in a population of transfected bacteria or cells totaling at most one million and more with typically only a few thousand clones each harboring a different mutant gene. Selection takes advantage... 

Single cell-derived colonies or foci are readily analyzed by cell colony hybridization (Avraham et al., 1989). Although cell colony hybridization on animal cells was described early (Villarreal and Berg, 1977), the subsequently developed dot/slot blot hybridization is normally preferred. Colony hybridization can have some particular advantages, however, such as the quantification of transfected cells or foci, the isolation of certain foci or colonies and may be useful for screening of stable somatic cell hybrids. 

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