Mouse Ltk - cells were transfected with four different plasmids for autoinducible and highly-inducible expression of the bacterial lacZ gene and cultivated in suspension. Two selection genes, thymidine kinase (tk) and neomycin resistance (neo r), were used to select the clones in both cell lines. The resulting two cell lines, designated M4 and R2, differ in that the inducible MMTV promoter from mouse mammary tumor virus (MMTV) controls glucocorticoid receptor (gr) gene and lacZ gene expression in the M4 cell line ("autoinducible"), while the constitutive rous sarcoma virus (RSV) promoter controls gr gene expression and the MMTV promoter controls lacZ gene expression in the R2 cell line ("highly-inducible"). Both cell lines were stable with respect to reproducibility of growth rate in spinner flasks and inducibility of β-galactosidase expression. The exponential growth rate of R2 cells was slower than that of M4 cells before induction because the R2 cell line continuously expressed gr genes under the constitutive RSV promoter, and the percent reduction of exponential growth rate mainly caused by gr gene expression was about 20%, The inducibility of the M4 cell line was greater than that of the R2 cell line because in the M4 cell line MMTV promoter controlled gr and lacZ gene expression autoinducibly. Maximum induction of the M4 cell line occurred after induction with the hormone dexamethasone (Dex) at 10 -7 M, and the final β-galactosidase content increased 400-fold after induction. The optimum conditions for inducer concentration and induction time were determined, and the highest production of β-galactosidase occurred when Dex was added after the cell concentration had reached its maximum in batch culture. Dex (10 -9 M) is a critical inducer concentration in view of inducibility between M4 and R2 cell lines. The inducibility of R2 cell line is higher than that of the M4 cell line from 0 to 10 -9 M Dex, but the inducibility of M4 was higher than that of the R2 cell line at Dex concentrations of more than 10 -9 M.
|Number of pages||8|
|Publication status||Published - 1996 Dec 1|
ASJC Scopus subject areas
- Food Science