These results confirm that exon 3 is not properly spliced in 01640 mutants, the mRNA contains P-element sequences, and this likely produces a truncated form of the CF2 protein.
To exclude the possibility that protein translation and thus gfp-expression is not equivalent between ORF4−Tet+ mutant and parental or ORF4 revertant virus, we investigated the infection kinetics of the ORF4 mutant at the mRNA level by quantitative real-time RT-PCR.
As Figure 4A shown, under the non-stressed status, the Crh mRNA expression level in Per1 mutant mice was significant lower than the one in M-WT mice (F = 20.9, p<0.01), by contrast, in Per2 mutant mice the mRNA expression level was significantly increased (F = 14.6, p<0.05).
In female mutant mice, the mRNA levels of the above mentioned genes were not changed.
Interestingly, ptb and buk, encoding a phosphate butyryltransferase and a butyrate kinase, respectively, were upregulated in the spo0A mutant at the mRNA level.
Yeast TREX mutants show a nuclear export defect for bulk poly A+ RNAs and are synthetically lethal with many mutants of the mRNA machinery [ 30].
Prg4, a secreted glycoprotein, which is already known to be positively regulated by TGF-β in superficial zone chondrocytes [ 32] and has a protective effect on cartilage surfaces, was downregulated in the DNIIR mutants at the mRNA level.
Our data show that AONs targeting PE44.1 sequences can indeed modify the splicing pattern of the mutant mRNA, reducing the aberrantly spliced form that includes PE44.1 and concomitantly increasing the normal mRNA form.
Analysis of the potential secondary structure of the mutant mRNA in the translation initiation region suggested that the Ala1 (GCC) and Leu2 (CUG) codons used are likely to be involved in a hairpin structure with the Thr13 (ACG) and Gly14 (GGG) codons, hindering effective translation at the ribosome.
The stable expression of a shRNA targeting the mutant KrasG12D allele led to a decreased expression of the mutant allele at the mRNA level in CD18/HPAF-shKras cells.
