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Cell cycle analysis of foreign gene (β‐galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter
Author(s) -
Gu Man Bock,
Todd Paul,
Kompala Dhinakar S.
Publication year - 1996
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19960505)50:3<229::aid-bit1>3.0.co;2-i
Subject(s) - recombinant dna , biology , mouse mammary tumor virus , gene , gene expression , microbiology and biotechnology , virus , virology , genetics
The cell cycle dependency of foreign gene expression in recombinant mouse L cells was investigated. Two different recombinant mouse L cell lines having the glucocorticoid receptor‐encoding gene and the lac Z reporter gene were used in this study. The lac Z gene expression was controlled by the glucocorticoid‐inducible mouse mammary tumor virus (MMTV) promoter in both cell lines. In “M4” cells the gr gene was under the control of another MMTV promoter, but in “R2” cells it was under the control of the constitutive Rous sarcoma virus promoter. These normally attachment‐grown cells were adapted to suspension culture, and a dual‐laser flow cytometer was used to simultaneously determine the DNA and foreign protein (β‐galactosidase) content of single living cells. Expression of β‐galactosidase as a function of cell cycle phase was evaluated for cells in exponential growth without any addition of the glucocorticoid inducer, dexamethasone. Cell cycle positions in the S phase were estimated on the basis of DNA content per cell, and position in the G1 phase was estimated on the basis of cell size as measured by pulse‐width time of flight. The results showed that β‐galactosidase synthesis occurred through all cell cycle phases, but the expression rate in the G1 phase was much lower than that in the S and G2/M phases in both cell lines. On the basis of cell size analysis, β‐galactosidase expression in M4 cells (with autoinducible promoter) was found to be higher than that in R2 cells (with inducible promoter) during the G1 phase. © 1996 John Wiley & Sons, Inc.