I. The E. coli Lac Operator-Repressor System is Functional for Control of Gene Expression in Animal Cells. II. Molecular Cloning and Characterization of the Mouse Skeletal Muscle (α) Actin Gene

Author: Hu, Mickey ChienTsung

Year: 1988

Degree: Dissertation (Ph.D.)

Advisor: Dougherty, Dennis A.

Committee Members: Campbell, Judith L.; Dervan, Peter B.; Davidson, Norman R.; Dougherty, Dennis A.; Wold, Barbara J.

Option: Chemistry

DOI: 10.7907/2a59-9p43

Abstract

We have investigated the use of the E. coli lac operator-repressor system to regulate the expression of genes introduced into mammalian cells by gene transfer. We find that the bacterial lac repressor protein encoded in a suitable expression vector is synthesized in mammalian cells in culture, assembles into a tetramer, enters the nucleus to some extent, and represses expression of another gene that has one or several lac operator sequences inserted into any one of several sites in the promoter region of the gene. Derepression can be achieved by exposure of the cells to IPTG. From a practical point of view of an inducible genetic switch, this system confers an induction level of somewhere between 10- and 20-fold in most of the cases we have tested. That is not better than those that have been achieved with heat shock, mouse mammary tumor virus, and metallothionein promoters. There may, however, be some situations and some promoters for which the use of the lac operator system is advantageous. We have also shown that this lac control system can be used to regulate the expression of genes introduced into Xenopus oocytes by micro-injection.

At present, we have been trying to revise this system by using a newly developed promoter containing a symmetric lac operator sequence inserted at the various strategic points within the human metallothionein IIA promoter and enhancer regions, which consist of several positive control elements, in order to achieve induction ratios of a factor of 100 or more. We have also generated a new lacI gene which encodes a repressor containing at its carboxyl terminus the nuclear localization signal of the SV40 large-T antigen. Overall, by combining the newly developed lac control promoters with the new repressor producing cell lines, we hope to generate an inducible expression system with large induction ratios that can be used as a general genetic switch in the future.

In section II, the nucleotide sequence of the mouse skeletal muscle (α) actin gene is presented and discussed.

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