Alteration of cellular sialic acid species by transfection of CMP-Neu5Ac hydroxylase cDNA

 Subculture target cells in 6-cm dish the day before transfection.

 Dilute 4 μg of plasmid DNA into 250 μL of Opti-MEM.

 Dilute 10–20 μL of Lipofectamine reagent into 250 μL of Opti-MEM.

 Combine diluted DNA and Lipofectamine reagent, and incubate for 15 min at room temperature.

 Replace culture medium with 2 mL of Opti-MEM.

 Add combined DNA-Lipofectamine complex (500 μL) to the cells.

 Incubate the cells at 37˚C for 1–5 h.

 Add 2 mL of complete medium containing serum dropwise.

 Incubate at 37˚C overnight.

 Replace the culture medium to fresh medium and culture at 37˚C. Go to Method 3 (Detection of Neu5Gc).

 Subculture packaging cells (e.g. Plat-A for human cells, Plat-E for rodent cells) in 10-cm dish the day before transfection.

 0.5 h prior to transfection, replace culture medium with 10 mL of fresh DMEM containing 4.5 g/L glucose (70% confluence at this point).

 Dilute 30 μg of plasmid DNA (Cmah cDNA cloned into MSCV (mouse stem cell virus) vector plasmid) to 876 μL with sterile water and add 124 μL of 2M Calcium Solution (BD CalPhos Mammalian Transfection Kit).

 Add DNA-calcium solution dropwise to 1 mL of 2X HBS with constant bubbling.

 Incubate the transfection solution at room temperature for 20 min to form calcium crystal conjugated with DNA.

 Gently vortex the transfection solution.

 Add the transfection solution dropwise to packaging cells.

 Incubate at 37˚C.

 Remove medium 6 h after transfection and add 4 mL of fresh medium.

 Incubate at 37˚C for 24 h.

 Collect and filter culture supernatant which contains retrovirus. Alliquot and store the virus at −80°C until infection.

 Add 4 mL of fresh medium to the cells and incubate for another 24 h.

 Collect and filter culture supernatant. Directly proceed to infection step or store the virus at −80°C.

 Add polybrene (6 μg/mL) to the retrovirus-containing culture supernatant and incubate for 10 min on ice.

 Add retrovirus to target cells (e.g. 500 μL/well in 24-well plate).

 Spin-infect (2,500 rpm (1,120 × g), 90 min, 32˚C).

 Add fresh medium (500 μL/well in 24-well plate) and incubate at 32˚C overnight.

 Add fresh medium and incubate at 37˚C. Go to Method 3 (Detection of Neu5Gc).

 Harvest the cells 48 h after transfection.

 Wash twice with PBS.

 Resuspend the cell pellet in 2 M acetic acid.

 Hydrolyze sialic acids for 2 h at 80˚C.

 Centrifuge (15,000 rpm, 5 min, 4˚C).

 Collect the supernatant.

 Recover cleared sialic acids using 3K cut off membrane (Microcon YM-3).

 Add equal volume of 2X DMB solution.

 Incubate for 2 h at 55˚C.

 Dilute with distilled water.

 Detect the peak of sialic acid by HPLC (Excitation wave length: 373 nm, Emission wave length: 448 nm).