Omozygous knock-in (LTR9NS/ LTR9NS) animals. The effect on Nras

Omozygous knock-in (LTR9NS/ LTR9NS) animals. The effect on Nras mRNA levels was highest in the spleen, where homozygous knock-in animals showed a fourfold increase in Nras mRNA relative to wild type (wt). Western blotting using an NRAS specific antibody detected higher protein levels in knock-in than in wt animals, again more pronounced in spleen than in HDAC-IN-3 chemical information thymus (Figure 3B). The liver samples were excluded from the Western analysis due to a low signal to noise ratio. Expression of the LTR-Nras chimeric transcripts previously identified in the tumor buy AKT inhibitor 2 harboring a provirus at position 9 [7] was verified by the RT-PCR using LTR and Nras specific primers, and it was confirmed that the generation of these transcripts does not abolish transcription from the normal Nras promoter (Figure 3C). Analysis of Nras mRNA levels in mice harboring the LTR9NAS allele with the LTR placed in the opposite transcriptional orientation of Nras revealed downregulation of Nras mRNA in spleen, thymus, and liver when analyzed with the amplicon spanning exons 2 and 3 (Figure 4a, upper panels). The mRNA levels in heterozygotes were intermediate between those of wt and of LTR9NAS/LTR9NAS animals. The largest effect was an about two-fold reduction observed in spleen tissue. In contrast, analysis using the amplicon spanning exons 6 and 7 detected an upregulation in animals harboring the LTR9NAS allele in spleen, but not in thymus and liver tissues. Western blotting analysis (Figure 4B) showed a decrease in NRAS protein in mice harboring the LTR9NAS allele in spleen and thymus in consistency with the mRNA levels detected with the exon2-exon3 probe.mouse knock-in models using retroviral sequences. These animals contain a single Akv1-99 LTR integrated at the exact same position as a previously identified retroviral integration in a tumor, placed in either the same or opposite transcriptional orientation relative to Nras and with or without a flanking floxed PGK/Tn5 neo cassette. We here report that the different alleles up or downregulate Nras expression to various degrees dependent upon the orientation and position of the LTR. Mice of this series have already proven valuable in the analysis of mechanisms of deregulation of host genes by insertional mutagenesis [8] as well as investigation of phenotypic effects of Nras over-expression [9].Results Generation of Alleles with Targeted Knock-in of an LTR and a Floxed PGK/Tn5 Neomycin CassetteThe positions chosen for targeted insertion of an LTRcontaining cassette corresponded to the three retroviral integrations identified in B-cell lymphomas by Martin-Hernandez et al. [7] within an 800 bp window upstream of the coding region for NRAS (Figure 1A). Integration 3 was located in the 39untranslated region of Csde1 upstream of the Nras promoter, whereas integrations 9 and 11 were both located in intron 1 of Nras. All three integrations had the same transcriptional orientation as Nras. To address the role of the orientation of the LTR, targeted insertions were made with the LTR in the same as well as the opposite transcriptional orientation as Nras. The knock-in plasmids harbored an Akv1-99 LTR and, to allow selection, a floxed PGKneomycin-resistance expression cassette placed in the same transcriptional orientation as the LTR. The alleles with the neomycin selection marker (neo) and an LTR in sense orientation relative to Nras are termed LTR3NS, LTR9NS, and LTR11NS for the three positions, respectively, whereas the alleles with neo a.Omozygous knock-in (LTR9NS/ LTR9NS) animals. The effect on Nras mRNA levels was highest in the spleen, where homozygous knock-in animals showed a fourfold increase in Nras mRNA relative to wild type (wt). Western blotting using an NRAS specific antibody detected higher protein levels in knock-in than in wt animals, again more pronounced in spleen than in thymus (Figure 3B). The liver samples were excluded from the Western analysis due to a low signal to noise ratio. Expression of the LTR-Nras chimeric transcripts previously identified in the tumor harboring a provirus at position 9 [7] was verified by the RT-PCR using LTR and Nras specific primers, and it was confirmed that the generation of these transcripts does not abolish transcription from the normal Nras promoter (Figure 3C). Analysis of Nras mRNA levels in mice harboring the LTR9NAS allele with the LTR placed in the opposite transcriptional orientation of Nras revealed downregulation of Nras mRNA in spleen, thymus, and liver when analyzed with the amplicon spanning exons 2 and 3 (Figure 4a, upper panels). The mRNA levels in heterozygotes were intermediate between those of wt and of LTR9NAS/LTR9NAS animals. The largest effect was an about two-fold reduction observed in spleen tissue. In contrast, analysis using the amplicon spanning exons 6 and 7 detected an upregulation in animals harboring the LTR9NAS allele in spleen, but not in thymus and liver tissues. Western blotting analysis (Figure 4B) showed a decrease in NRAS protein in mice harboring the LTR9NAS allele in spleen and thymus in consistency with the mRNA levels detected with the exon2-exon3 probe.mouse knock-in models using retroviral sequences. These animals contain a single Akv1-99 LTR integrated at the exact same position as a previously identified retroviral integration in a tumor, placed in either the same or opposite transcriptional orientation relative to Nras and with or without a flanking floxed PGK/Tn5 neo cassette. We here report that the different alleles up or downregulate Nras expression to various degrees dependent upon the orientation and position of the LTR. Mice of this series have already proven valuable in the analysis of mechanisms of deregulation of host genes by insertional mutagenesis [8] as well as investigation of phenotypic effects of Nras over-expression [9].Results Generation of Alleles with Targeted Knock-in of an LTR and a Floxed PGK/Tn5 Neomycin CassetteThe positions chosen for targeted insertion of an LTRcontaining cassette corresponded to the three retroviral integrations identified in B-cell lymphomas by Martin-Hernandez et al. [7] within an 800 bp window upstream of the coding region for NRAS (Figure 1A). Integration 3 was located in the 39untranslated region of Csde1 upstream of the Nras promoter, whereas integrations 9 and 11 were both located in intron 1 of Nras. All three integrations had the same transcriptional orientation as Nras. To address the role of the orientation of the LTR, targeted insertions were made with the LTR in the same as well as the opposite transcriptional orientation as Nras. The knock-in plasmids harbored an Akv1-99 LTR and, to allow selection, a floxed PGKneomycin-resistance expression cassette placed in the same transcriptional orientation as the LTR. The alleles with the neomycin selection marker (neo) and an LTR in sense orientation relative to Nras are termed LTR3NS, LTR9NS, and LTR11NS for the three positions, respectively, whereas the alleles with neo a.