Journal: Molecular Therapy. Methods & Clinical Development
Article Title: Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles
Figure Lengend Snippet: MS2-driven RNA packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of DNAse-treated supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
Article Snippet: In total, 4 µl RNA treated with TURBO DNAse (2 U/µl) was used for cDNA synthesis with the Superscript First-strand Synthesis System (Life Technologies).
Techniques: Plasmid Preparation, Construct, Positive Control, Derivative Assay, Expressing, Luciferase, Sequencing, Transmission Assay, Electron Microscopy, Negative Staining, Microscopy, Quantitative RT-PCR, Transfection, Amplification, Concentration Assay