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1.
Figure 7.

Figure 7. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Analysis of adenosine deamination of MosIR RNA. Adenosine deamination was analyzed by cloning RT–PCR products from the MosIR region indicated on the top. Results of editing analysis of MosIR transcripts from transgenic kidney and liver and MosIR-transfected 293 cells are shown below. Each row of squares represents one sequenced clone. Each square represents an individual adenosine in the analyzed sequence. A part of the PCR product is derived from the stem (dsRNA) sequence (white squares) and a part from single-stranded sequence (gray squares). A/G conversions are indicated by black squares.

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
2.
Figure 8.

Figure 8. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Analysis of adenosine deamination of MosIR-derived siRNAs. (A) Distribution of putatively edited SOLiD-identified small RNAs originating from EGFP and Mos inverted repeat (MosIR) sequences in 293 cells. Small RNAs are sorted along the X-axis according to their length (17–26 nt).The Y-axis shows the number of clones with EGFP- (upper graph) or MosIR-derived sequences (lower graph). The gray portion of each column indicates the fraction of siRNAs carrying up to four A/G sequence changes. (B) Putative editing is most pronounced in SOLiD-identified siRNA-like RNAs derived from the MosIR sequence. The graph shows A/G mismatch frequency found in the total population of small RNAs and 20–24 nt long RNAs derived from EGFP or MosIR sequences in 293 cells.

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
3.
Figure 3.

Figure 3. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Deep sequencing of MosIR-derived small RNAs. (A) Deep sequencing of small RNAs from brain using SOLiD technology. Absolute counts of the most abundant miRNAs and 20–23 nt RNAs derived from the EGFP coding sequence (EGFP) and from the Mos inverted repeat (MosIR) are shown on the left. The graph shows distribution of 17–25 nt small RNAs derived from the Mos inverted repeat. (B) Deep sequencing of small RNAs from 293 cells transfected with MosIR plasmid using SOLiD technology. Data are organized as in panel A. (C) Positions of putative MosIR siRNAs along the MosIR hairpin. Graph depicts cumulative counts of 5′-ends of putative MosIR-derived siRNAs (20–24 nt long reads) found in the transgenic brain and 293 cells transiently transfected with the MosIR transgene.

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
4.
Figure 4.

Figure 4. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

MosIR has no effect on transcriptional silencing of cognate sequences. (A) A schematic composition of the MosP reporter transgene. (B) EGFP signal in MosP-positive and WT newborn and adult mice. (C) EGFP fluorescence in tails of mice carrying MosP and MosIR transgenes is not reduced. Bright signal in some MosP-positive tails (e.g. first on the left in the top row) is caused by the lack of pigmentation of the tails. The MosP tail on the left in the lower row belongs to an F1 animal where we observed a spontaneous transcriptional silencing accompanied by DNA methylation of the MosP promoter (Supplementary Figure S4). All images were obtained using the same settings. (D) EGFP signal in tail, kidney, brain and oocytes of MosP, MosIR and MosP & MosIR mice. Organs were collected from siblings of one litter and their images were taken with the same camera settings.

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
5.
Figure 6.

Figure 6. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Microarray analysis of HeLa and 293 cells expressing MosIR. Microarray analysis of HeLa and 293 cells transfected with MosIR plasmid (MosIR) or pCAGEGFP plasmid (control). (A) A hierarchical clustering of microarray samples using probe sets differentially expressed (>2-fold change) between MosIR and control samples in both lines shows good concordance of duplicates and clustering according to the cell type. This suggests that there is not a common set of genes induced by MosIR in these two cell lines. (B) A minimal overlap of differentially expressed genes in HeLa and 293 cells. Genes corresponding to commonly up-regulated probe sets are listed in Supplementary Table SI. (C) MA plots of HeLa and 293 transcriptome change upon MosIR expression document small and distinct changes in hybridization intensities of probe sets. The X axis shows Affymetrix probe hybridization signal intensities (log2 average expression); the Y axis, shows relative changes in probe intensity between MosIR and control samples. Each point represents one probe set. Red and blue color highlight probe sets significantly up-regulated and down-regulated in the other cell line. (D) Absence of dsRNA-induced transcriptome signature in HeLa and 293 cells expressing long dsRNA. Genes previously identified as genes induced by dsRNA treatment are shown (18).

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
6.
Figure 5.

Figure 5. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Analysis of the IFN response genes in mice and cell lines. (A) Analysis of expression of IFN stimulated genes (ISGs) Ifit1, Oas1, Pkr and Ddx58 in different tissues of MosIR mice. The expression was estimated by qPCR and is shown relative to wild-type siblings. For each gene, samples from three MosIR animals were analyzed in duplicates. Hprt was used as an internal standard. Error bars = SEM. (B) Analysis of expression of ISGs in 293 cells transfected with 50 and 500 ng (per well in a 24-well plate, the total amount of the transfected DNA was kept constant by adding pCAGEGFP DNA) of the MosIR plasmid 48 h after transfection. The expression was estimated by qPCR and is shown relative to 293 cells transfected with 100 ng/well of the pCAGEGFP plasmid. For comparison, ISGs were stimulated by adding poly I:C to the media. HPRT1 was used as an internal standard. The experiment was performed in triplicates. Error bars = SEM. (C) Western blot analysis of induction of PKR phosphorylation by the MosIR plasmid. Cells in 6-well plates were transfected with 100, 500 or 1800 ng of the MosIR plasmid and harvested for western blot analysis 24 h after transfection. The total amount of the transfected DNA was adjusted to 2 µg in all samples by adding corresponding amount of pCAGEGFP DNA. The majority of transfected cells showed green fluorescence (data not shown), toxicity of the MosIR to the cells was not apparent. Poly I:C was added to the media to a final concentration of 1 µg/ml and it was apparently toxic to the cells.

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
7.
Figure 2.

Figure 2. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Absence of RNAi in somatic cells. (A) A schematic composition of the Mos3 reporter transgene. (B) EGFP signal in different organs of MosIR, Mos3 and Mos3 & MosIR mice. Organs were collected from siblings of one litter. All images were taken with the same settings. The exposure of was shorter than in Figure 1C in order to obtain non-saturated brain EGFP signal. (C) qPCR analysis of different tissues shows little, if any, RNAi in somatic tissues and a strong RNAi effect in oocytes. Mos3 reporter expression in Mos3 & MosIR mice is shown relative to its expression in Mos3 mice. Samples were collected from two Mos3 and two Mos3 & MosIR animals and analyzed by qPCR in triplicates. Hprt was used as an internal normalization standard. Tissue heterogeneity could contribute to the lower level of Mos3 in the kidney. Error bars = SEM. (D) MosIR does not induce RNAi when transfected to somatic cells. The 293 cells stably transfected with a non-targeted firefly luciferase (FF) reporter and a targeted Renilla luciferase (RL) reporter (carrying Mos sequence in its 3′-UTR, Supplementary Figure S1B) were grown in 24-well plates and transfected with increasing amounts of the MosIR plasmid. pCAGEGFP was added to transfection mixtures to balance different amounts of the MosIR plasmid and to maintain the total amount of transfected DNA constant. Cells were harvested 48 h after transfection and luciferase activity was analyzed. Both luciferase activities are shown relative to cells transfected with 0 ng of the MosIR plasmid. NT = non-transfected cells. Transfection efficiency estimated by microscopic examination of EGFP fluorescence was >90%. Error bars = SEM.

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.
8.
Figure 1.

Figure 1. From: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Analysis of MosIR expression and phenotype. (A) Schematic composition of the MosIR transgene. (B) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. (C) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. (D) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. (E) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). (F) qPCR of Mos mRNA in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. (G) RNase T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).

Jana Nejepinska, et al. Nucleic Acids Res. 2012 January;40(1):399-413.

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