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. 2019 Feb 13;47(8):e44. doi: 10.1093/nar/gkz093

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Gene editing activities of various LVLPs. (A) Effects of MS2 aptamers on SaCas9 mRNA level. Plasmid DNA expressing SaCas9^nxMS2 (250 ng) was co-transfected into GFP-reporter cells grown in 24-well plates with plasmid DNA expressing HBB sgRNA1 (250 ng). SaCas9 mRNA level was compared by RT-qPCR with HBB sgRNA1 as a control. *** and ### indicate P < 0.001 when SaCas9^0xMS2 was compared with SaCas9^1xMS2, and when SaCas9^1xMS2 was compared with SaCas9 with more than one MS2 aptamer. (B) Effects of MS2 aptamers on SaCas9 gene editing activity. GFP-reporter cells were transfected as in A. Flow cytometry was performed 72 h after transfection. *** indicates P < 0.001 when SaCas9^0xMS2 was compared with SaCas9 with at least one MS2 aptamer. (C) Examining the effects of aptamer numbers on LVLP gene editing activity. LVLP-containing supernatants (titer determined by p24 ELISA) were co-transduced with 50 ng HBB sgRNA1-expressing IDLV into 2.5 × 10⁴ GFP-reporter cells. Flow cytometry was performed 72 h after transduction. ‘a’ indicates that 45 ng p24 of SaCas9^1xMS2 LVLPs obtained significantly higher GFP-positive rate (P < 0.001) than all other LVLPs at the same dosage; ‘b’ indicates that 45 ng p24 of SaCas9^12xMS2 LVLPs obtained significantly lower GFP-positive rate (P < 0.001) than SaCas9^2xMS2 or SaCas9^3xMS2LVLPs; ‘c’ indicates that 15 ng p24 of SaCas9^12xMS2 LVLPs obtained significantly lower GFP-positive rate (P < 0.05) than all other LVLPs at the same dosage. Each data point is the mean of three replicates. (D) Flow cytometry analysis of GFP-positive cells generated after transducing various SaCas9-containing LVLPs. Designated amounts of SaCas9-containing LVLPs were co-transduced with 60 ng p24 HBB sgRNA1-expressing IDLV into 2.5 × 10⁴ GFP-reporter cells. Each point was the average of indicated replicates (numbers in parentheses). * indicates P < 0.05 when SaCas9^1x^MS²-HBB 3′ UTR LVLP was compared with any other particles of the same dosage; *** indicates P < 0.001 when LVLPs generated without ABP and aptamers were compared with any other particles of the same dosage. (E) RT-qPCR comparisons of SaCas9 mRNA copy numbers in different types of LVLPs. RNA was purified from lentivirus or LVLPs containing 30 ng p24. 30 ng p24 of GFP-lentivirus was added in each sample for experimental control. Copy numbers were compared to normal lentiviral vectors known to contain two RNA genomes/LV particle. Shown are individual data points and mean ± SEM. ns, no significant difference; ***P < 0.001 compared with any other groups. For A, B and E, Tukey's multiple comparison tests were performed following ANOVA analysis. For C and D, Bonferroni posttests were performed following two-way ANOVA.