A new generation of pPRIG-based retroviral vectors

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.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.Retroviral vectors are widely used to stably express any protein of interest. They potentially can transduce a large variety of primary or immortalized cells types, either in vitro, ex vivo or in vivo. Selection of transduced cells is generally accomplished by the co-expression of a fluorescent protein, a membrane protein or a gene encoding a drug-resistance. The more the expression of the two proteins correlates, the more the relevant cells in the selected population will be enriched. One way to obtain coupled expression in cells is to express both proteins from a single bicistronic mRNA: the 5'