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A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

Figure 4

Overview of the lentiviral (A) or retroviral (B) vectors for protein expression or depletion.

Entry vectors can be recombined using the LR clonase into the Destination vectors connected by arrows. For RNAi-mediated depletion, miRNA-based and shRNA-based vectors are available for either constitutive or inducible depletions (see Figure 2A for details). For expression, various tags are available (see Figure 2B for details). The RNAi cassettes can be inserted in the middle of the lentiviral backbone (pLenti X1 series), in the 3′ LTR (pLenti X2 series) or in a backbone expressing GFP (pLenti CMV GFP). For RNAi studies using retroviruses, all the Destination cassettes have been inserted in the 3′ LTR of the vectors. For protein expression, the EF-1α, PGK or CMV promoters are available for lentiviruses. CMV-driven constructs can be either constitutive or inducible. For the retroviral vectors, no constitutive expression of cDNAs can be attained in T-REx cell lines using the pQCXI series vectors because they all contain the inducible CMV/TO promoter. Similarly, the drug resistance gene will be repressed in a T-REx cell line since it is after an IRES element under the control of the CMV/TO promoter. However, the pQCXP CMV/TO can be used for inducible expression of cDNAs under constitutive puromycin selection in T-REx cell lines. See Table 2 for details.

Figure 4