Figure 1.
miRNA-based regulation of TetR-KRAB control of transgene expression.
(a) Schematic representation of the miRNA-TetR-KRAB regulatory and reporter cassettes. (b) In the absence of tissue-specific miRNA, the repressor is translated and is free to bind the tetO operator, thus preventing transgene expression. However in the presence of a tetracyclin analog (Doxycycline) the repressor does not bind and transcription is activated.(c) In targeted cells tissue-specific miRNA binds to its complementary target and results in degradation of the TetR-KRAB mRNA. As a result, transgene expression occurs.
Figure 2.
Schematic diagram of the recombinant lentivirus vectors used to accomplish liver-specific transgene expression.
(a) & (b) Lentiviral vector encoding four target sequences of miR-122, TetR-KRAB and GFP under the control of a mTTR liver specific promoter (a) or constitutively active CMV promoter (b). (c) Lentiviral vector without target sequences of miR-122 and expressing GFP from liver-specific mTTR promoter. (d). Inducible TetR-KRAB lentiviral vector system that includes a GFP gene under control of a CAG promoter as part of a bicistronic unit comprising the KRAB based repressor (PLVCT). cPPT, central polypurine tract; IRES, internal ribosomal entry site; WPRE, woodchuck hepatitis virus post-transcriptional element; RRE: Rev protein responsive element; pA, polyadenylation site; CMV, cytomegalovirus; CAG, CMV immediate enhancer/β-actin; PGK, phosphoglycerate kinase; mTTR,murine liver-specific transthyretin receptor promoter.
Figure 3.
miR-122 concentrations and reporter gene expression in transduced cells.
(a) miR-122 expression levels in Huh7 and 293T cells detected by RT-qPCR. Three independent experiments were performed in duplicate. *Statistically significant differences P = 0.03 (two-tailed, unpaired Student’s t-test). (b) & (c) GFP expression in transduced Huh7 and 293T cells. After 10 days of culture, with or without doxycycline, transduced 293T (b) or Huh7 (c) cells were analyzed for GFP expression by FACS. Results are expressed as a percentage of MFI. In the TetR-KRAB system, the expression of GFP is optimal in the presence of doxycycline and corresponds to 100% of the MFI. The raw data corresponding to 100% are indicated above each histogram bar. (d) RT-qPCR of GFP mRNA normalized to values obtained for 18S RNA. Data shown are mean and error bars indicate the standard deviation (SD) of 3 independent experiments performed in triplicate. (e) Representative images of GFP expression in cells at 10 days after transduction. Lentiviral vectors encoding TetR-KRAB mRNA with four target sequences for miR-122 and GFP gene under the control of a liver specific promoter in 293T and Huh7 cells with or without doxycycline treatment. Magnifications ×50. (f) Representative high power fields showing immunofluorescence staining of TetR-KRAB (red) in Huh7 cells transduced with recombinant lentiviruses. Four target sequences for miR-122 were absent (left panel) or present (right panel) in the TetR-KRAB-expressing cassettes of the vectors, which also produced GFP (green) constitutively. Magnifications ×400.
Figure 4.
Lentiviral vectors used to target transgene expression to macrophage-derived cells.
(a) Schematic diagram of the lentivirus vectors used to target cells of immune lineage. (b) miR-142 expression levels in Huh7, 293T and NR8383 cells detected by RT-qPCR. Three independent experiments were performed in duplicate. * Statistically significant differences P = 0.043 (two-tailed, unpaired Student’s t-test). (c) & (d) Huh7, 293T and NR8383 cells were transduced with two different lentiviral vectors carrying the TetR-KRAB sequence follow by 4 copies of miR-142 target (c) or miR-122 (d). After 10 days of culture with or without doxycycline, transduced cells were analyzed for GFP expression by FACS. Results are expressed as a percentage of mean fluorescence intensity (MFI). In TetR-Krab system, the expression of GFP is optimal in the presence of doxycycline, corresponding to 100% of the MFI. The raw data corresponding to 100% are indicated above each histogram bar.Data shown are mean and error bars indicate the SD of 3 independent experiments performed in triplicate.
Figure 5.
Lentiviral vectors used to target transgene expression to skeletal muscle-derived cells.
(a) Schematic diagram of the lentivirus vectors used to accomplish skeletal muscle-specific expression. (b) miR-133 expression levels in C2C12d, C2C12ud and 293T cells detected by RT-qPCR. Three independent experiments were performed in duplicate. *Statistically significant differences P = 0.0185 (two-tailed, unpaired Student’s t-test). (c) Differentiated or undifferentiated C2C12 cells (C2C12d and C2C12ud respectively) were transduced with lentiviral vectors carrying the TetR-KRAB sequence followed by four copies of miRNA target of miR-122 or miR-133. After 10 days of culture with or without doxycycline, transduced cells were analyzed for GFP expression by FACS. Results are expressed as a percent of MFI. The raw data corresponding to 100% are indicated above each histogram bar. Data shown are mean and error bars indicate the SD of 3 independent experiments performed in triplicate.
Figure 6.
Assessment of efficacy of miRNA-regulated TetR-KRAB control of transgene expression in vivo.
(a) Schematic diagram of the AAV vectors used for in vivo studies. (b) GFP fluorescence was measured in live animals at 8 weeks after injection with recombinant AAVs. Mice received AAVs encoding the TetR-KRAB sequence without copies of miRNA target located downstream. Animals did or did not receive doxycycline in their drinking water. The color scale next to the images indicates the signal intensity. (c) & (d) Results at 8, 12 and 16 weeks post injection are expressed in counts/mm2/second. Data shown are mean and error bars indicate the SD. * Statistically significant differences P<0.05 (two-tailed, unpaired Student’s t-test), n = 4 per group.