Pcdna3.1 ct gfp topo

The mouse skeletal muscle cell line (myoblast) C2C12 (ATCC, Manassas, VA) was used as the cell system for this study due to the relatively prominent chromocenters and low endogenous MeCP2 expression22 (link). In order to express MeCP2 in a mammalian system, the full length MECP2_E1 gene was amplified using reverse transcriptase polymerase chain reaction followed by cloning into the expression construct pcDNA3.1 CT-GFP-TOPO (placing GFP at the C-terminal end of MeCP2), according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA). Mutations were introduced through PCR based site-directed mutagenesis according to the manufacturer’s instructions (QuikChange Lightning Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA), in order to generate the MeCP2 missense substitutions R106W, R111G, N126I, R133L, R133C, A140V, P152A, P152R, P152H, F157I, T158M, also an N-terminal missense substitution A2V, and a C-terminal deletion P390_P391del, for use as positive controls, in addition to the wild type (WT) MeCP2.

Coding sequences of PcSOD3.1, PcSOD3.2, PcSOD1, and PcYellow-like were amplified from a cDNA pool of all tissues using Pfx-Polymerase (Invitrogen, Thermo scientific). The fragments were cloned into pIB/V5-HIS TOPO vectors (Invitrogen, Thermo scientific), which is lacking T7-promotor sites, interfering with dsRNA synthesis. For that, this part of each coding region has been chosen, which resulted to be unique after off-target-prediction88 (link). These fragments were amplified using PCR with primers containing both a 5′-T7-promotor-sequence. Also, the unique part of the coding sequence of the green fluorescent protein from pcDNA3.1/CT-GFP-TOPO (Invitrogen, Thermo scientific) was subjected to the described protocol and cloned into a pIB-vector. The cloning products were primarily amplified using E. coli TOP10F’ cells (Invitrogen, Thermo scientific) and sequenced, to confirm unaltered dsRNA templates.