Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice.
BACKGROUND
Precise control of transgene expression is essential for a variety of applications ranging from gene-function analysis, biopharmaceutical manufacturing to next-generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene-therapy-based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease.


METHODS
We have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide-responsive promoter consisting of a central operator module (ETR) specific for the macrolide-dependent transactivator (ET1) is flanked by two minimal promoters (P(hCMVmin); P(hsp70min)) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels.


RESULTS
Bidirectional expression configurations enabled excellent macrolide-adjustable coregulation profiles of two secreted reporter genes or one-vector-based autoregulated fine-tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO-K1 cell derivatives transgenic for macrolide-controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses.


CONCLUSIONS
Based on their in vivo compatibility, the versatile bidirectional and macrolide-responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality.
Fux
Cornelia
C
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland.
Weber
Wilfried
W
Daoud-El Baba
Marie
M
Heinzen
Christoph
C
Aubel
Dominique
D
Fussenegger
Martin
M
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004921:Erythropoietin / Q000096:biosynthesis; D005260:Female; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D018942:Macrolides / Q000378:metabolism*; D051379:Mice; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D023822:Tissue Engineering; D019076:Transgenes
D018942:Macrolides; D004921:Erythropoietin; D000469:Alkaline Phosphatase