Deregulated Nras expression in knock-in animals harboring a gammaretroviral long terminal repeat at the Nras/Csde1 locus

Abstract

To investigate mechanisms and phenotypic effects of insertional mutagenesis by gammaretroviruses, we have developed mouse lines containing a single Akv 1-99 long terminal repeat (LTR) and a floxed PGK/Tn5 neomycin cassette at the Nras proto-oncogene at positions previously identified as viral integration sites in Akv 1-99 induced tumors. The insert did not compromise the embryonic development, however, the cassette had an effect on Nras expression in all tissues analyzed. Cre-mediated excision of the PGK/Tn5 neomycin cassette in two of the lines caused upregulation of Nras. Altogether, the knock-in alleles are characterized by modulation of expression of the target gene from more than ten-fold upregulation to three-fold downregulation and exemplify various mechanisms of deregulation by insertional mutagenesis. LTR knock-in mice may serve as a tool to investigate mechanisms of retroviral insertional mutagenesis and as a way of constitutive or induced modulation of expression of a target gene.

Figure 1. Overview of knock-in alleles.

(A). Schematic representation of Nras. Arrows indicate the identified Akv 1­99 proviral integrations (integration 3, 9 and 11) . 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.

Figure 2. Analysis of Nras expression in knock-in clones of mouse ES cells.

The analysis included two LTR3NS clones, five LTR3NAS clones, two LTR9NS clones, three LTR9NAS clones, three LTR11NS clones, one LTR11NAS clones as well as parental CJ7 cells. Nras mRNA was quantified by qPCR employing an amplicon covering part of exon 2 and exon 3 (insert). Expression was normalized to that of Tbp and represented as relative to the parental CJ7 ES cell line. The panels below the histogram present Western blot analysis of NRAS in protein extracts from the listed ES cell clones. GAPDH was used as reference.

Figure 3. Analysis of knock-in animals harboring the LTR integrated in the sense orientation at position 9.

(A). Nras 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 Tbp or Gapdh 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 Nras 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 Nras intron. LTR initiated transcription does not seem to suppress the activity of the normal Nras promoter, as the putative Nras transcript could be detected in both wild type and homozygous LTR9NS animals employing the appropriate Nras specific primers (right half of gel).

Figure 4. Analysis of knock-in animals harboring the LTR integrated in the antisense orientation at position 9.

(A). Nras 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 Tbp or Gapdh 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 Nras 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.

Figure 5. Nras expression in knock­in animals with and without the neomycin selection marker.

Nras 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 Tbp or Gapdh 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.

Figure 6. Analysis of knock-in animals harboring the LTR inserted at position 3.

Nras expression was quantified by qPCR employing an amplicon 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 Tbp or Hprt 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. Alleles with the cassette in sense (panel A) or antisense (panel B) orientation were analyzed. Paired Student’s t test was used to determine p-values relative to +/+ animals.