Analysis of knock-in animals harboring the LTR integrated in the sense orientation at position 9.

<p>(A). <i>Nras</i> expression was quantified by qPCR employing two different methods, SYBR green (amplicon covering part of exon 2 and 3) or a TaqMan hydrolysis probe (amplicon covering part of exon 6 and 7). Expression was normalized to that of <i>Tbp</i> or <i>Gapdh</i> depending on the employed strategy (SYBR green or TaqMan probe, respectively) and represented as relative to that of wild type animals. N represents the number of animals in the different groups. Paired Student’s t test was used to determine p-values relative to +/+ animals. (B). Western blot analyses of spleen and thymus samples using antibodies against NRAS or GAPDH. C) PCR analysis of mRNA from spleen of homozygous LTR9NS (samples 1 and 2) and wild type animals (samples 3 and 4). Two distinct chimeric mRNAs can be detected by an LTR and an <i>Nras</i> specific primer in combination (left half of gel). These transcripts depicted at the bottom of the figure contain viral as well as cellular sequences and differ in length due to splicing or not from a cellular splice donor at the first <i>Nras</i> intron. LTR initiated transcription does not seem to suppress the activity of the normal <i>Nras</i> promoter, as the putative <i>Nras</i> transcript could be detected in both wild type and homozygous LTR9NS animals employing the appropriate <i>Nras</i> specific primers (right half of gel).</p

<i>Nras</i> overexpression resulted in weight reduction and early lethality.

<p>(A). LTR insertion at the proviral integration site in the <i>Nras</i> gene in transcriptional (sense) orientation. The LTR of Akv1–99 was inserted before nucleotide 1062 of the GenBank sequence no. L19607. Boxes indicate exons, coding regions in black. Exons and introns are not depicted in scale. Arrow indicates proviral integration. (B). Frequency of different genotypes in offspring from heterozygous matings. Numbers in parenthesis indicate the expected number of mice of each genotype. (C). Relative <i>Nras</i> expression in different tissues of young mice normalized to the expression of wild type tissues. <i>Gapdh</i> was used as internal standard. Error bars indicate standard deviation. Numbers under bars indicate numbers of analyzed mice. (D). Weight analysis of young mice. Homozygous animals showed a reduced weight gain after one week. Error bars indicate standard deviation. An average of 8 <i>LTR/LTR</i> mice, 12 <i>LTR/+</i> mice, and 5<i>+/+</i> mice are shown however, not all mice contribute to all time points. (E). Analysis of spleen weight relative to body weight. Bars show average of relative spleen weight normalized to wild type littermates. Relative spleen weight = Total spleen weight/total body weight. <i>LTR/LTR</i> mice have a lower relative spleen weight compared to wild type mice (n = 9).</p

Flow cytometry analysis of myeloid cells in thymus of young <i>Nras^LTR9S</i> mice.

<p>(A). Percentage of thymic cells within region 1 normalized to the population gated in the same way as shown for spleen data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042216#pone-0042216-g002" target="_blank">Figure 2</a>. Cell frequency was significantly higher in <i>LTR/LTR</i> mice compared to wt. (B). Percentage of thymic cells stained with CD11b and Gr-1 markers within region 1 normalized to the total cell population. The Gr-1^dimCD11b⁺ population was upregulated in <i>LTR/LTR</i> mice compared to wt (data were log transformed to obtain variance homogeneity). (C). Percentage of thymic cells stained with CD11b and Gr-1 markers within region 2 normalized to the total cell population. The Gr-1⁺CD11b⁻ population was upregulated in <i>LTR/LTR</i> mice compared to wt (data were log transformed to obtain variance homogeneity). (D). Percentage of thymic cells stained with CD11b and Gr-1 markers within region 3 normalized to the total cell population. The Gr-1⁺CD11b⁻ population was upregulated in <i>LTR/LTR</i> mice compared to wt. Dots represent measures of individual mice.</p

Analysis of knock-in animals harboring the LTR integrated in the antisense orientation at position 9.

<p>(A). <i>Nras</i> expression was quantified by qPCR employing two different methods, SYBR green (amplicon covering part of exon 2 and 3) or a TaqMan hydrolysis probe (amplicon covering part of exon 6 and 7). Expression was normalized to that of <i>Tbp</i> or <i>Gapdh</i> depending on the employed strategy (SYBR green or TaqMan probe, respectively) and represented as relative to that of wild type animals. N represents number of animals in the different groups. Paired Student’s t test was used to determine p-values relative to +/+ animals. (B). Western blot analyses of spleen and thymus samples using antibodies against NRAS or GAPDH. (C). Rapid amplification of cDNA ends: Initiation sites of alternative transcripts within the <i>Nras</i> gene or viral LTR were identified by the usage of the GeneRacerTM kit (Invitrogen). Position of the detected transcription start sites are depicted with respect to the first nucleotide of exon 1 or 4. Height of the bars indicates the frequency of the detected transcripts.</p

<i>Gscf</i> and <i>Gfi1</i> mRNAs were upregulated in young <i>Nras^LTR9S</i> mice.

<p>(A). Q-RT-PCR quantification of <i>Gcsf</i> expression in spleen normalized to <i>Hprt</i> expression. Compared to wt <i>Gcsf</i> was upregulated in <i>LTR/LTR</i> spleen. (B). Q-RT-PCR quantification of <i>Gfi1</i> expression in spleen normalized to <i>Tbp</i> expression. Compared to wt <i>Gfi1</i> was upregulated in <i>LTR/LTR</i> spleen. (C). Q-RT-PCR quantification of <i>RasGRP1</i> expression normalized to <i>Gapdh</i> expression. Compared to wt <i>RasGRP1</i> was upregulated in <i>LTR/LTR</i> spleen. (D). Q-RT-PCR quantification of <i>Sos1</i> expression normalized to <i>Gapdh</i> expression. Compared to wt <i>Sos1</i> was upregulated in <i>LTR/LTR</i> spleen. For A and B three mice of each genotype were analyzed, for C and D, 3 LTR/LTR, 7 LTR/+, and 3+/+ mice were analyzed. Error bars indicate standard deviation.</p

<i>Nras</i> expression in knock­in animals with and without the neomycin selection marker.

<p><i>Nras</i> expression was quantified by qPCR employing two different methods, SYBR green (amplicon covering part of exon 2 and 3) or a TaqMan hydrolysis probe (amplicon covering part of exon 6 and 7). Expression was normalized to that of <i>Tbp</i> or <i>Gapdh</i> depending on the employed strategy (SYBR green or TaqMan probe, respectively) and represented as relative to that of wild type animals. Panels A and B: qPCR and Western analysis of the LTR9S allele. Only +/+ and +/LTR9S animals are included since LTR9S/LTR9S animal die within three weeks. Panels C and D: qPCR and Western Blot analysis of the LTR9AS allele. Paired Student’s t test was used to determine p-values relative to +/+ animals.</p

Overview of knock-in alleles.

<p>(A). Schematic representation of <i>Nras</i>. Arrows indicate the identified Akv 1­99 proviral integrations (integration 3, 9 and 11) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056029#pone.0056029-MartinHernandez1" target="_blank">[7]</a>. Boxes represent exons and the coding region is depicted in black. (B). Representation of the “targeting cassettes” introduced in the sense (S) and antisense (AS) knock-in models. Upon expression of Cre recombinase a LoxP sequence (triangle) and the neomycin selection marker (Neo) can be removed from the construct. LTR = long terminal repeat.</p

Flow cytometry analysis of the spleen of young <i>Nras^LTR9S</i> mice.

<p>One representative analysis of a wild type and <i>LTR/LTR</i> mouse is shown. Debris and dead cells were excluded by applying a FSC threshold (channel 180). Significant upregulation of SSC high granular cells was detected in <i>LTR/LTR</i> mice (population is marked as region 1). A second population within the monocytic region is marked as region 2. The lymphocyte region is marked as region 3. Each region was analyzed for CD11b and Gr-1 stain.</p

Analysis of granular cells in the blood of young <i>Nras^LTR9S</i> mice.

<p>Relative numbers of granular and monocytic cells in blood smears. More granular cells and correspondingly less monocytic cells were found in <i>LTR/LTR</i> mice compared to wt (p<0.004).</p

Flow cytometry analysis of myeloid cells in spleen of young <i>Nras^LTR9S</i> mice.

<p>(A). Percentage of splenocytes within region 1 normalized to the gated population defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042216#pone-0042216-g002" target="_blank">Figure 2</a>. Cell frequency was significantly higher in <i>LTR/LTR</i> mice compared to wt. (B). Percentage of splenocytes within region 2 normalized to the gated population defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042216#pone-0042216-g002" target="_blank">Figure 2</a>. The population was significantly smaller in <i>LTR/LTR</i> mice compared to wt. (C). Percentage of splenocytes stained with CD11b within region 1 normalized to the total cell population. The CD11b⁺ population was significantly larger in <i>LTR/LTR</i> mice compared to wt. (D). Percentage of splenocytes stained with CD11b and Gr-1 within region 1 normalized to the total cell population. The Gr-1^dimCD11b⁺ population was increased in <i>LTR/LTR</i> mice compared to wt. (E). Percentage of splenocytes stained with CD11b and Gr-1 within region 2 normalized to the total cell population. Dots represent individual mice.</p