Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-6

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>en unaffected controls (C1 – C7), two -mutant males with congenital encephalopathy (CE) and five females diagnosed with RTT, with or without identified mutations. The mutant samples are identified by their brain bank numbers. expression was normalized to , a constitutive ribosomal protein gene that is expressed at a similar level in brain. To calculate relative fold changes, control sample C4 was arbitrarily set at 1. All samples were run in triplicate on the same plate

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-2

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p> J-allele mice, while B-allele mice are on a C57BL/6 background. The effects of array batch, and to a lesser extent, age, are also apparent. We, therefore, compared only microarrays from littermates, at the same age and using the same array batch, in our search for differentially expressed genes

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-0

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>tched: coding sequence. Grey: non-coding parts of transcript. Exons 1 and 2 that are alternatively spliced, and intron 2, are not drawn to scale. Horizontal bar above exon 4 denotes location of the amplicon analyzed by qRT-PCR (in Figure 1B) The (J-allele) mutant has a deletion of exon 3 that encodes the N-terminal half of the MBD. In the (B-allele) mutant, exon 3, the coding portion of exon 4 and part of the 3'UTR are deleted. This larger deletion eliminates ~ 97% of the coding sequence. Using primers within the deleted region, transcript was undetectable in mutant mice with the B-allele, when four wild-type and four mutant samples were compared at 8 wk. This proves the specificity of the primers. In mice with the J-allele, mean expression was higher in the mutants, but this difference was not statistically significant (= 0.11), when six wild-type and seven mutant samples were compared at 8 wk

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-5

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>e M, 100 bp ladder; lane 1, -mutant chromatin precipitated with anti-MeCP2 (negative control); lane 2, wild-type chromatin precipitated with no antibody present; lane 3, wild-type chromatin precipitated with rabbit IgG, lane 4: wild-type chromatin precipitated with anti-MeCP2, lane 5: input DNA, lane 6: no DNA control. The precipitated DNA was amplified with primers specific for the promoter regions of and , and for the promoter/DMR of . The Gtl2-B amplicon is located within the Gtl2-A amplicon. Precipitation with anti-MeCP2 shows enrichment relative to the IgG and no antibody controls, indicating that MeCP2 is associated with the designated regions . As negative controls, PCR was performed using primers covering regions that do not interact with MeCP2 for each ChIP reaction (not shown)

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-1

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>heir wt sibs rather than with mutants of other litters. Litters are identified by A – F. The array of the litter B wild-type sib did not pass quality control standards

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-7

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>tched: coding sequence. Grey: non-coding parts of transcript. Exons 1 and 2 that are alternatively spliced, and intron 2, are not drawn to scale. Horizontal bar above exon 4 denotes location of the amplicon analyzed by qRT-PCR (in Figure 1B) The (J-allele) mutant has a deletion of exon 3 that encodes the N-terminal half of the MBD. In the (B-allele) mutant, exon 3, the coding portion of exon 4 and part of the 3'UTR are deleted. This larger deletion eliminates ~ 97% of the coding sequence. Using primers within the deleted region, transcript was undetectable in mutant mice with the B-allele, when four wild-type and four mutant samples were compared at 8 wk. This proves the specificity of the primers. In mice with the J-allele, mean expression was higher in the mutants, but this difference was not statistically significant (= 0.11), when six wild-type and seven mutant samples were compared at 8 wk

And were selected for validation of expression changes by qRT-PCR analyses in B-allele mice

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p> Cerebellum microarray expression data (M) are compared with qRT-PCR data from cerebellum (RC), forebrain (RF) and hippocampus (RH). For RC of and , the 8 wk samples from the microarray analysis were used. RC2 data are derived from an independent set of litters at 7.5 to 8 wk. Error bars represent SEM. One asterisk indicates < 0.05, two asterisks indicate < 0.001. expression was markedly increased in the cerebellum of 8 wk B-allele mice on microarray (2-fold, = 0.011, four mutant/wild-type pairs) and qRT-PCR (3.5 fold, < 0.001, four mutant/wild-type pairs) as well as in the forebrain of a different set of mice at 7.5 wk (1.9 fold, < 0.05, three mutant/five wild-type). expression was elevated 1.4-fold on microarrays (< 0.05, five mutant/wild-type sibs) and qRT-PCR (RC) = 0.053, four mutant/wild-type sibs). The RC2 data (five mutant/three wild-type) were more variable and yielded = 0.16. displayed an ~ 1.5 fold increase in expression on microarrays (= 0.03, four mutant/wild-type pairs) and qRT-PCR (= 0.06, five mutant/wild-type pairs) at 2 wk (RC) and was not changed in hippocampus (RH). On microarray, expression was increased at 8 wk, but by qRT-PCR we found highly variable expression in cerebellum (RC and RC2). expression was not significantly increased (= 0.29, eight mutant/eight wild-type) in the forebrain

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-3

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>decreased (0.8-fold or lower) at < 0.05 are included. Transcripts in the overlap regions are described in Additional file . The same criteria as in Figure 4A were applied. . The 39 transcripts include the 15 overlapping DEG from Figure 4B as well as genes that were significantly changed at one age in one mutant and at another time point in the other mutant. The list is provided in the Additional file