( ow basal expression and high induction ...)pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins

ow basal expression and high induction ratios are affected by copy number. Hence, viral infection (at an optimal MOI) of the target cell should yield a high frequency of clones capable of mediating desirable expression profiles without exhaustive colony screening.

We describe the vector pFB-ERV, an MMLV-based replication-defective retroviral vector for delivery of the ecdysone receptor proteins RXR and VgEcR.

Vector Description

Fig.1

The vector pFB-ERV contains a tricistronic message transcribed from the CMV promoter (Figure 1). The receptor proteins VgEcR and RXR are expressed from the first and second open reading frames (ORF), respectively,

and the neomycin-resistance gene is expressed from the third ORF. Translation of the RXR and Neo ORFs is mediated by the EMCV-IRES (both IRESs are identical). In this context, G418 selection and maintenance of the expression cassette in stably infected cells ensures that the receptor-encoding mRNA is transcribed. The CMV expression cassette was built into a self-inactivating (SIN) vector backbone,^6,7 in which the retroviral promoter within the U3 region of the 3 LTR was deleted. In SIN vectors, viral genomic RNA is expressed from the 5 viral LTR in packaging cells; however, upon infection the virus replicates in such a way that the (inactive) 3 U3 promoter sequences are transferred to the 5 LTR, and the proviral 5 promoter is then inactive in infected cells. The rationale for this construction is the following: The CMV promoter is stronger than the MMLV LTR and is persistently active in a wider range of cell types, and inactivation of the 5 LTR obviates potential interference with the CMV promoter; the CMV promoter can be readily replaced with a cell-type promoter of interest using the unique EcoR I and Fse I sites without concern of ubiquitous read-through fr
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