Effects of gene therapy on muscle 18S rRNA expression in mouse model of ALS

Abstract

Background: The efficiency of gene therapy experiments is frequently evaluated by measuring the impact of the treatment on the expression of genes of interest by quantitative real time PCR (qRT-PCR) and by normalizing these values to those of housekeeping (HK) genes constitutively expressed throughout the experiment. The objective of this work was to study the effects of muscle gene therapy on the expression of 18 S ribosomal RNA (Rn18S), a commonly used HK gene.

Findings: Mouse model of motor neuron disease (SOD1-G93A) was injected intramuscularly with Brain-derived neurotrophic factor (BDNF-TTC) encoding or control naked DNA plasmids. qRT-PCR expression analysis was performed for BDNF and HK genes Rn18 S, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and β-actin (Actb). We report that elevated BDNF expression in the injected muscle was accompanied with increased Rn18 S expression, whereas Gapdh and Actb were not affected. Increased "ribosomal output" upon BDNF stimulation was supported by increased steady-state levels of ribosomal protein mRNAs.

Conclusions: Ribosomal RNA transcription may be directly stimulated by administration of trophic factors. Caution should be taken in using Rn18 S as a HK gene in experiments where muscle metabolism is likely to be altered by therapeutic intervention.

Figure 1

Up-regulation of Rn18 S upon BDNF-treatment. Gene expression analysis in muscle tissue of SOD1-G93A animals intramuscularly injected with pcDNA3.1-CMV control plasmid (dark grey bars) or pcDNA3.1-CMV-BDNF-TTC plasmid (light grey bars) at ten days (age 70 days) or fifty days (age 110 days) post-injection. A) BDNF expression is unchanged (P = 0.258) at age of 70 days but shows 2.4-fold increase (P = 0.007) in therapeutically treated animals at age of 110 days. B) Rn18 S expression is not altered at age of 70 days (P = 0.298) but is 1.8-fold higher (P = 0.009) at age of 110 days. C) Actb and D) Gapdh expression is unaltered in all conditions (P > 0.05). Error bars indicate standard error of mean. Symbol ** denotes statistical significance level P ≤ 0.01.

Figure 2

Up-regulation of ribosomal protein mRNAs upon BDNF-treatment. Gene expression analysis was carried out as in Figure 1. pcDNA3.1-CMV control plasmid -treated muscles are shown as dark grey bars and pcDNA3.1-CMV-BDNF-TTC plasmid treated muscles as light grey bars. A) Transcripts for ribosomal proteins Rps13, Rps17, Rpl41 and Rpl44 were unaffected (P = 0.854, P = 0.472, P = 0.735 and P = 0.522, respectively) by BDNF-treatment at age of 70 days. B) Transcripts for ribosomal proteins were increased by BDNF-treatment at age of 110 days: Rps13 (8.6 fold, P < 0.01), Rps17 (13.1-fold, P < 0.01), Rpl41 (17.4-fold, P < 0.01) and Rpl44 (7.1-fold, P < 0.05). Error bars indicate standard error of mean. Symbols ** and * denote statistical significance level P ≤ 0.01 and P ≤ 0.05, respectively.