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. 2019 Apr 18;47(13):e74. doi: 10.1093/nar/gkz267

Figure 2.

Figure 2.

CRISPRai delivered by baculovirus activated Sox9 and repressed PPAR-γ in stem cells. (A) Baculovirus vectors and targeting sites. Bac-aS expressed four sgRNAa targeting four positions of rat Sox9 promoter. Bac-iP expressed four sgRNAi targeting four positions of rat PPAR-γ gene. Bac-aS-iP expressed four Sox9-targeting sgRNAa and four PPAR-γ-targeting sgRNAi. All these three baculoviruses contained two loxP sites to flank the CRISPRai module and sgRNA. Bac-Cre expressed Cre recombinases. (B, C) Sox9 and PPAR-γ expression. rBMSC and rASC were mock-transduced (Mock group) or transduced with Bac-aS, Bac-iP or Bac-aS-iP at MOI 300 and cultured in α-MEM. The expression levels were measured by qRT-PCR at 3 dpt and normalized to those of Mock group. (D, E) Kinetics of Sox9 and PPAR-γ expression. rBMSC were mock-transduced (Mock group), singly transduced with Bac-aS-iP (MOI 300) or co-transduced with Bac-aS-iP and Bac-Cre at MOI 300/100 and cultured in α-MEM. The expression levels were measured by qRT-PCR at 3 dpt and normalized to those of Mock group. The data represent means± SD of three independent culture experiments. ****P < 0.0001. *** P < 0.001. ** P < 0.01. *P < 0.05. ns, not significant (P > 0.05).