A new generation of pPRIG-based retroviral vectors

Background: Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES- containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/ or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer- promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site ( MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild- type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS ( pPRIG HAa/b/c). Results: The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG- based vectors ( collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS ( 3' HA PRIGs); ii) a functional domain ( ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS ( << modular >> PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R ( << single color/ resistance >> PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES- eGFP, IRES- eCFP or IRES- Puro- R containing PRIGs, respectively ( << dual color/ selection PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells. Conclusion: These novel vectors allow for straightforward detection of any expressed protein ( 3' HA PRIGs), for functional studies of chimeric proteins ( << modular >> PRIGs), for multiple transductions and fluorescence analyses of transduced cells ( << single color/ resistance >> PRIGs), or for quantitative detection of studied proteins in independently identified/ selected transduced cells ( << dual color/ selection >> PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.