Mouse work was performed in the Biomedical Sciences Unit at the University of Oxford as authorized by the UK Home Office (Animal Scientific Procedures Act 1986). Taiwanese SMA mice were bred and maintained as described on The Jackson Laboratory website and as described previously.36 (link),63 (link) MEFs were isolated from strain FVB.Cg-Smn1tm1HungTg(SMN2)2Hung/J (Jackson Laboratory; 005058), crossed with strain FVB.129P2(B6)-Smn1tm1Hung/J (Jackson Laboratory; 031678), using a method described previously.64
After 2 additional days of culturing in MEF culture medium, the cells were plated for ASO transfection. For the MEF lines with sufficient cell counts, duplicate wells were plated (one for transfection with the 5′ UTR ASO and one for transfection with the NTC ASO). Single wells were plated for the MEF lines with fewer cells. The MEFs were transfected with ASO using RNAi MAX as described above. 3 days later, the MEFs were collected in RIPA buffer and subsequently assayed by immunoblotting.
KO/D7;SMN2- and KO/F7-immortalized MEFs37 (link) shared by the Burghes lab were transfected with ASO and immunoblotted 2 days post-transfection.
After 2 additional days of culturing in MEF culture medium, the cells were plated for ASO transfection. For the MEF lines with sufficient cell counts, duplicate wells were plated (one for transfection with the 5′ UTR ASO and one for transfection with the NTC ASO). Single wells were plated for the MEF lines with fewer cells. The MEFs were transfected with ASO using RNAi MAX as described above. 3 days later, the MEFs were collected in RIPA buffer and subsequently assayed by immunoblotting.
KO/D7;SMN2- and KO/F7-immortalized MEFs37 (link) shared by the Burghes lab were transfected with ASO and immunoblotted 2 days post-transfection.
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