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. 2021 Nov 20;23:582–592. doi: 10.1016/j.omto.2021.11.012

Figure 3.

Figure 3

Knocking out PKR, OAS1, and LDLR in 293T cells additively increased titer, RNA, and physical particles

(A) Fold difference of titers of Lenti/βAS3-FB packaged in single, double, and triple-KO cells (n = 12 dishes of identical cultures from four independent experiments; bars represent mean with SD; unpaired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). The double-KO isogenic clones were created by electroporating RNP targeting the OAS1 gene in the PKR−/− isogenic clones and selecting an isogenic clone with OAS1 and PKR knocked out. The triple-KO isogenic clones were created by electroporating RNP targeting LDLR gene in the PKR and OAS1 double-KO clone and selecting an isogenic clone with OAS1, PKR, and LDLR knocked out. (B and C) The absolute quantification and percentage of complete vRNA in Lenti/βAS3-FB viral particles measured by ddPCR (n = 9 dishes of identical cultures from three independent experiments; bars represent mean with SD; unpaired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (D) Concentrations of physical particles in Lenti/βAS3-FB unconcentrated virus quantified by p24 ELISA (n = 9 dishes of identical cultures from three independent experiments; bars represent mean with SD; unpaired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (E) Titers of Lenti/βAS3-FB, PYC-CAR, EFS-ADA, and Mini-G packaged in parental 293T and OAS1, PKR, and LDLR KO cells (n = 9 dishes of identical cultures from three independent experiments; bars represent mean with SD; unpaired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).