Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

COS cells were grown in 100-mm culture dishes in a humidified incubator at 37°C in 5% COz with DME media containing 0.1 fcg/ml penicillin and streptomycin and 10% FCS (DME/10% FCS) . Confluent cell monolayers were trypsinized and split 1 :8 two days before transfection . The large EcoRI fragment from clone MII-8 containing the entire Man II open reading frame was excised and ligated into the EcoRI site of the pXM COS cell expression vector (58) . Recombinant plasmids were checked by restriction mapping to confirm the correct orientation of the insert. COS cells (80% confluent) were transfected either with the pXM without an insert, pXM containing the Man II insert in the correct orientation (MII-pXM), or pXM containing the Man II insert in the antisense orientation (asMII-pXM) by liposomemediated transfection (Lipofectin ; Bethesda Research Labs, Gaithersburg, MD) as described by the manufacturer. Briefly, 20 kg of the relevant plasmid were mixed with 50 A,1 of Lipofectin reagent in 3 ml of serum-free medium (Opti-MEM ; Bethesda Research Labs) and incubated at room temperature for 15 min. Monolayer cultures were rinsed twice with Opti-MEM and incubated with the 3 ml media containing the plasmid/lipofectin mixture tbr 6-8 h at 37°C. The cultures were rinsed twice with DME/10 % FCS and incubated with DME/10% FCS at 37°C for 24-72 h . Cells prepared for inrmunofluorescence studies were plated on coverslips placed in 24-well rnicrotiter plates, grown to 50 % confluency, and transfected at the same plasmid/Lipofectin/Opti-MEM ratio as in the larger scale transfections (total volume of 200 pl/well) .