Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR-0

<p><b>Copyright information:</b></p><p>Taken from "Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR"</p><p>http://www.molecularneurodegeneration.com/content/2/1/16</p><p>Molecular Neurodegeneration 2007;2():16-16.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018688.</p><p></p>NA preparations of the four genotypes (KO male: y/- ; WT male: y/+; Heterozygote female: +/- ; WT female: +/+) were submitted to the different PCR amplification protocols detailed in the methods section, before loading on a 1.5% agarose gel

Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR-1

<p><b>Copyright information:</b></p><p>Taken from "Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR"</p><p>http://www.molecularneurodegeneration.com/content/2/1/16</p><p>Molecular Neurodegeneration 2007;2():16-16.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018688.</p><p></p>NA preparations of the four genotypes (KO male: y/- ; WT male: y/+; Heterozygote female: +/- ; WT female: +/+) were submitted to the different PCR amplification protocols detailed in the methods section, before loading on a 1.5% agarose gel

Mutant forms of MeCP2 that cause RTT retain their repressive effect on MHC class I expression.

<p>Panel A: Schematic representation of the MeCP2 protein. Red and orange arrows indicate the positions of the mutations introduced in MeCP2 by site-directed mutagenesis (MBD: methyl-CpG binding domain, TRD: transcription repression domain, WW: group II WW-domain-binding region). Panel B: N2A cells transfected with empty pcDNA3.1 (mock cells), with pcDNA3.1 expressing Myc-tagged MeCP2<i>A</i> (pMeCP2<i>A</i>-<i>myc</i>) or with pcDNA3.1 expressing Myc-tagged MeCP2<i>A</i> with the R133C point mutation (pMeCP2<i>A</i>-R133C-<i>myc</i>) were stained with mouse anti-Myc 9E10 monoclonal antibody, and FITC-labelled anti-mouse IgG antibody. Coverslips were mounted in DAPI-containing ProLong Gold antifade reagent (Molecular Probes) before observation by fluorescence microscopy. Panel C: N2A cells transfected as in panel B were double immunostained for cell surface MHC class I and intracellular Myc-tagged MeCP2, then analysed by flow cytometry. Similar data were obtained for all four mutated forms of MeCP2<i>A</i> and MeCP2<i>B</i> (not shown), and these observations were reproduced in three independent transfection experiments.</p

MeCP2 overexpression diminishes MHC class I expression.

<p>N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (K^k, L^d or D^d), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2<i>A</i> (grey fill) and 12 independent transfections with vectors expressing human MeCP2<i>A</i> (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2<i>A</i> or <i>B</i> isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, <i>p</i><0.01 ; ***, <i>p</i><0.001).</p

Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.

<p>N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule L^d or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001).</p

Evaluation of MHC class I expression in adult mouse brain slices.

<p>Serial frozen sections of adult male wild-type and MeCP2^−/y littermates were analysed for expression of MHC class I by immunohistochemistry using the rat R1-21.2 monoclonal antibody and EnVision detection technology (Dako). For the negative control, the same staining process was used omitting the primary antibody. Similar results were obtained with the M1/42 monoclonal antibody. Similar results were obtained in independent experiments on brains from three different pairs of mice.</p

Deletion of MECP2 does not affect basal or IFN-γ -induced MHC class I expression in primary cultures of mixed glial cells.

<p>Mixed glial cell cultures established from two-day old wild-type, MeCP2^+/− and MeCP2^−/y mice (10, 6 and 8 animals per group, respectively) were treated or not with IFN-γ on the second day of culture and analysed two days later by flow cytometry for MHC class I expression. Neurons were identified by their intracellular staining with an anti-β-III-tubulin antibody (inset). Large cells, containing mainly astrocytes, were analysed separately by an appropriate forward/side scatter gate. Primary spleen fibroblasts from the same mice were also subjected or not to IFN-γ treatment and stained for their MHC class I expression. Panel A: Representative histograms showing cell surface staining (x axis) against cell number (y axis), obtained with cells from wild-type and MeCP2^−/y male littermates. White-filled curves represent background staining, gray-filled curves represent MHC I-specific staining. Panel B: MHC class I fold-induction in response to IFN-γ was calculated as the ratio of MFI of treated cells (induced MHC I level) on MFI of untreated cells (basal MHC I level). Grey-filled squares show MHC class I fold-induction for individual mice and for each cell type. White-filled squares represent the group's mean of fold-inductions (±SD).</p