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Published on: June 25, 2015
Translational modulation of proteins expressed from bicistronic vectors.
Prasun J Mishra1, Lata G Menon, Pravin J Mishra
1Department of Pharmacology, Robert Wood Johnson Medical School, Cancer Institute of New Jersey, USA
Molecular Imaging
|December 17, 2009
Summary
This study introduces a novel method using dihydrofolate reductase (DHFR) and methotrexate (MTX) to enhance bicistronic vector expression. This approach allows for controlled, long-term induction of two proteins from a single promoter.
Area of Science:
- Molecular Biology
- Gene Expression Systems
Background:
- Bicistronic vectors enable co-expression of two genes from one promoter.
- A common limitation is reduced expression of the second gene product.
- Dihydrofolate reductase (DHFR) and methotrexate (MTX) have been previously used to modulate protein levels.
Purpose of the Study:
- To overcome the limitation of reduced second cistron expression in bicistronic vectors.
- To explore the use of DHFR complementary DNA in bicistronic vectors for inducible protein expression.
- To develop a method for modulating the levels of two proteins expressed from a bicistronic construct.
Main Methods:
- Engineered bicistronic vectors encoding DHFR complementary DNA.
- Induced protein expression using methotrexate (MTX) treatment.
- Utilized rapamycin pretreatment followed by MTX treatment for modulation.
- Evaluated protein induction levels in vitro and in vivo.
Main Results:
- DHFR partner protein in bicistronic constructs showed prolonged induction compared to endogenous DHFR or DHFR fusion proteins upon MTX treatment.
- A strategy combining rapamycin pretreatment and MTX treatment allowed for cap-independent modulation of two protein levels.
- Demonstrated successful induction and modulation of protein levels in DHFR-based bicistronic constructs.
Conclusions:
- DHFR-based bicistronic vectors offer a viable strategy for enhanced and controlled co-expression of two proteins.
- MTX treatment provides a method for sustained induction of the second cistron product.
- Combined rapamycin and MTX treatment enables fine-tuning of protein levels from bicistronic vectors.
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