Abstract
BackgroundRetroviral 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).ResultsThe 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.ConclusionThese 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.
Highlights
Retroviral vectors are valuable tools for gene transfer
The pAP2 plasmid [1,2] belongs to this kind of retroviral vector since it contains eGFP as a selectable marker just downstream of the IRES element from the EMCV
PAP2 allows a potent CMV-driven expression upon transient transfection while retrotranscription restores a complete, Long Terminal Repeat. ECMV (LTR)-controlled, pro-viral sequence. Despite this improvement, the pAP2 vector suffers from two major flaws: 1) its multicloning site (MCS) is limited; and 2) its IRES sequence is weak as a result of the creation of a HindIII site in its 3' terminus, which corresponds to two point mutations with respect to the original EMCV sequence
Summary
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 allows a potent CMV-driven expression upon transient transfection while retrotranscription (and integration) restores a complete, LTR-controlled, pro-viral sequence Despite this improvement, the pAP2 vector suffers from two major flaws: 1) its multicloning site (MCS) is limited; and 2) its IRES sequence is weak as a result of the creation of a HindIII site in its 3' terminus, which corresponds to two point mutations with respect to the original EMCV sequence. These mutations, though widely found in IRES containing vectors, destroy the last IRES ATG, normally used by the ECMV virus [2]
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