Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences

Area of Science:

• Molecular Biology
• Gene Expression

Background:

• Bicistronic constructs allow co-expression of multiple genes from a single transcript.
• Internal ribosomal entry sites (IRES) are commonly used to drive cap-independent translation of downstream open reading frames.
• Short synthetic intercistrons offer an alternative for linking genes in bicistronic vectors.

Purpose of the Study:

• To evaluate the efficiency of second gene translation in bicistronic constructs using a short synthetic intercistron versus IRES.
• To assess the stability and expression of bicistronic constructs with different gene combinations.

Main Methods:

• Constructing bicistronic vectors with Herpes simplex virus-thymidine kinase (HSV-TK)/neomycin (NEO) or human glucocerebrosidase (hGC)/methotrexate (MTX) resistant dihydrofolate reductase (DHFR) genes.
• Utilizing a 36-bp synthetic intercistron or IRES to link genes.
• Transfection and retroviral infection of mammalian cells.
• Assessing gene expression via drug resistance assays and enzyme activity measurements.
• Analyzing proviral DNA and RNA integrity.

Main Results:

• Second gene translation was efficient with both IRES and the 36-bp intercistron upon initial transfection.
• Retroviral expression of HSV-TK/NEO bicistronic constructs with the 36-bp intercistron showed intact gene expression.
• However, hGC/DHFR bicistronic constructs with the 36-bp intercistron experienced loss of the hGC coding region in infected cells.
• IRES-linked hGC/DHFR constructs maintained expected function and expression.

Conclusions:

• Short synthetic intercistrons can facilitate efficient second gene translation in bicistronic constructs.
• The utility of short synthetic intercistrons is dependent on the specific gene combination and construct stability.
• IRES elements may offer greater reliability for certain gene pairings due to improved construct integrity.