Control of gene expression in mammalian cells
We have a general interest in the control of gene expression and a particular interest in immunoglobulin production. A major project at present is aimed at the utilisation of the high secretory capacity of myeloma cells in the expression of transfected genes and, hence, the development of a useful mammalian host/vector system.
To this end we have isolated some unusual myeloma cell transfectants which secrete novel proteins in particularly high yield. These clones contain small numbers of copies of the vector sequences integrated at a single site in the genome and we are examining the sites of chromosomal integration in order to understand their unusual activity. Cloning of the flanking regions may allow us to identify novel control regions involved in gene expression.
We are also developing the use of specific recombinases to target ingoing DNA to these high expression sites.
References
- Harrison TM, Hudson K, Munson SE, Uff S, Glassford S. (1994). Biochim. et Biophys. Acta 1260, 147-156. "Derivation and partial analysis of two highly active myeloma cell transfectants"
Intracellular sorting of regulated secretory proteins
We expect the control of expression in transfected myeloma cell lines to be principally at the transcriptional level. However, we are also interested in post-translational control of expression particularly in terms of transfer and processing through the secretory pathway.
Myeloma cells secrete proteins in a constitutive manner (ie. continuously) whereas other cell lines (principally of exocrine or endocrine origin) possess a regulated secretory pathway whereby certain proteins are segregated into specialised storage granules which are only released from the cell in a stimulus-dependent manner. Using transfection of manipulated gene sequences encoding such proteins, we are trying to understand how they are recognised and sequestered from other proteins in the secretory pathway and thereby targetted to the secretory granules.
References
- Chidgey MAJ, Harrison TM (1990). Eur. J. Biochem. 190, 139-144. "Renin is sorted to the regulated secretory pathway in transfected PC12 cells by a mechanism which does not require expression of the pro-peptide"
- Harrison TM, Chidgey MAJ, Uff S (1996). Cell Biol. Int. 20, 293-300. "Novel markers for constitutive secretion used to show that tissue plasminogen activator is sorted to the regulated pathway in transfected PC12 cells"
