Figure 1. Distribution and levels of tTA mRNA and protein
A. Northern blot including olfactory epithelium (OE), spinal cord (SC) and brain (B)
extracts, showing two bands with the tTA probe in double transgenic mice. From a single
representative double transgenic line (997 EGFP-TRE-hG93ASOD1cDNA). B. Western
blot of protein distribution across areas in A (anti-tTA monoclonal; MobiTec, Göttingen,
Germany). Positive control: homogenates from the activator PrP-tTA 959 mouse line.
From the same representative transgenic line. C. Western blot for the nuclear localization
of the tTA protein across the nervous system areas. From the same representative
transgenic line. Numbers indicate protein molecular weights. D. Quantitative analysis of
EGFP and human SOD1 production related to tTA levels. To determine if the production
of responder elements was dose-dependent on tTA, activator and responder homozygous
genotypes were established for the 997 EGFP-TRE-hG93ASOD1cDNA line. To obtain
homozygous genotypes, double transgenic mice were crossed to activator PrP-tTA mice or
the corresponding responder line. Hemizygous littermates were used for comparison. The
genotypes were established by real-time PCR. Data are all from groups of 2-3 mice, and
experiments were replicated twice. The densiometric analysis of bands was performed
with a computer-assisted image analysis system (AIS 3.0-Imaging Research) All samples
were normalized to -actin levels. E. Northen and Western blots of representative samples
quantified in D. The responder EGFP and human SOD1 mRNA and/ or protein levels are
irrespective of tTA levels (observe right panel in E). This suggestes that non-limiting
theresholds caused the transcriptional impairments observed. AA, homozygous activator
genotypes; RR, homozygous responder gentotypes; Aa, hemizygous activator genotypes;
Rr, hemizygous responder genotypes; 1, littermates harbouring 25% to 50% (poor) FVB/N
strain content; 2, littermates harbouring 50% to 75% (enriched) FVB/N content; 3; double
transgenic mice with 50% FVB/N content, relative to the set of crosses to obtain the
homozygous genotypes.
Name
F.SOD2
R.SOD2
F.EGFP
R.EGFP
F.SOD1
R.SOD1
F.tTA
R.tTA
Primers for screenings and analysis of cDNA expression
Sequence
Anealing temperature
and size amplicon
5’-TGCACTGAAGTTCAATGGTGG-3’
56 °C; 238 bp
5’-TAGAGCAGGCAGCAATCTGT-3’
5’-ACCACATGAAGCAGCACGACT-3’ 60 °C; 490 bp
5’-CTTGTACAGCTCGTCCATGCC-3’
5’-GATTCCATGTTCATGAGTTTGG-3’
56 °C; 360 bp
5’-GGCCTCAGACTACATCCAAG-3’
5’-GAGCAGCCTACATTGTATTGG-3’
56 °C; 458 bp
5’-GGCCGAATAAGAAGGCTGG-3’
F-PCMV1
R-PCMV1
Primers for real-time PCR analyses
5’-AGGCGTGTACGGTGGGAGG-3’
60 °C; 150 bp
5’-CCCGGGTACCGAGCTCGAA-3’
F xist
R xist
5’-TGATACGGCTATTCTCGAGCC-3’
5’-TCAAGGCGAATCCCGCAACC-3’
F QtTA
R QtTA
5’-AGAGCAGCCTACATTGTATTGG-3’ 60°C; 99
5’-AAGGGCAAAAGTGAGTATGGTG-3’ control
F.tetSOD1
60 °C; 137 bp, GeneBank
NT_039711. Normalisating
purposes.
bp.
Internal
Primers for cloning
5’-CTGTGCAGTCCTCGGAACCAGGA-3’
R.tetSOD1 5’-AAGGAAAGAAGCTAGCAGGATAACAGATGAGTTAAGGG-3’
Table 1. Primer sequences.