Functional characterisation of MeCP2 mutations found in male patients with X linked mental retardation

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Abstract
MeCP2 cDNA bearing missense mutations were generated by site directed mutagenesis using mismatched primers as described previously.11 A full length MeCP2 cDNA12 was used as a template for PCR. Insert of the A140V5, 6 or E137G6 mutant DNA was cloned into the BspEI and XhoI sites of pEGFP-C1(Clontech), an enhanced fluorescence vector, and the EcoRI and BamHI sites of the Drosophila expression vector pAc5.1/V5-His (Invitrogen). Mouse L929 cells were transfected with GFP expression constructs using Superfect (Qiagen). Two days later, cells on chamber slides were fixed with 3.7% formaldehyde for 10 minutes, permeabilised with 0.5% Triton X-100 for 20 minutes, and counterstained with 4`,6-diamidino-2-phenylindole (DAPI) (Sigma). The specimens were observed under an Olympus fluorescence microscope using the appropriate optical filter. The transient expression analysis using Drosophila SL2 cells was performed as follows. The SNRPN-luciferase reporter construct13 was treated with SssI (CpG) methylase (New England Biolabs) in the presence of 5 mmol/l S-adenosylmethionine as described previously.14 A total of 1.4 × 105 SL2 cells were grown in 0.7 ml of Schneider's Drosophila medium in a 24 well plate. A total of 0.4 μg of the luciferase reporter construct was cotransfected with 0.2 μg of an Sp1 expression plasmid, pPacSp1, 0. 01 μg of pAc5.1-pRL,15 and various amounts of expression plasmid (1-100 ng) bearing genes encoding MeCP2 mutants into Drosophila SL2 cells by the calcium phosphate method. The total amount of transfected DNA was adjusted by adding pAc5.1/V5-His vector. After 48 hours, the cells were lysed with 100 μl of lysis buffer and 10 μl of lysate was assayed for firefly and Renilla luciferase activities using the Dual-Luciferase reporter assay system (Promega). All transient transfection assays were carried out at least three times independently.