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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 1. — Developing LVLPs for SaCas9 mRNA packaging. (A) MCP-viral protein fusion proteins and SaCas9-MS2 aptamer fusion RNA for packaging SaCas9 mRNA in LVLPs. MCP: MS2coat protein; VPR: viral protein R, NEF: negative regulatory factor; NC, nucleocapsid protein; MA: matrix protein. Dashed line indicates the deleted region in MA. (B) Plasmids for making lentivirus and LVLP. The aptamer-binging protein MCP and the MS2 aptamer are shown in red and green respectively. LTR: long terminal repeats; Ψ: lentivirus packaging signal. (C) Effects of MCP-fusion proteins on lentivirus production. 5 × 10⁵ cells in six-well plates were transfected with the indicated plasmids (5 μg total DNA). Twenty four hours after transfection the medium was replaced with 2 ml fresh medium and p24 was assayed after 24 h. Individual data points and mean ± SEM are shown. Only MA-MCP modification decreased virus production (P < 0.05). (D) Best gene editing activity was obtained when MCP was fused to NC. SaCas9^1xMS2 was expressed during LVLP production. 250 μl LVLP-containing supernatant and 250 μl IDLV-expressing HBB sgRNA1 were co-transduced into 2.5 × 10⁴ GFP-reporter cells. ** indicates P < 0.01 when LVLPs from NC-MCP fusion was compared with other particles; * indicates P < 0.05 compared with any other particles. (E) Flow cytometry analysis of GFP reporter cells transduced with particles made with or without NC-MCP modified packaging plasmid. Increasing volumes of supernatants were co-transduced with 50 ng of HBB sgRNA1-expressing IDLV into GFP reporter cells. When producing particles without packaging plasmid, the packaging plasmid was replaced with pKanCMV-mRuby3-10aa-H2B expressing mRuby. ***P < 0.001 when the GFP-positive rates of the two conditions were compared (Bonferroni posttests following ANOVA). (F) Plasmid DNA contributed little in generating GFP-positive reporter cells. pSaCas9^1xMS2 (first column) or pSaCas9^1xMS2-HBB sgRNA1 plasmid DNA (second column) was transfected into GFP reporter cells to observe GFP-positive cells. pSaCas9^1xMS2-HBB sgRNA1 was also used to make LVLPs to transduce into GFP reporter cells alone (100 ng p24, the third column) or with 50 ng p24 of IDLV expressing HBB sgRNA1 (the fourth column). *** indicates P < 0.001. For C, D and F, Tukey's multiple comparison tests were performed following ANOVA analysis.