Basic Research Of Biological Function Of GDP-fucose Transporter

GDP-fucose transporter transport GDP-fucose in which synthetize in the cytoplasm to the Golgi apparatus.Fucosyltransferase(Fut)catalyzes the fucosylation using GDP-fucose via 1,2?1,3/4?1,6-linkage in the Golgi apparatus in mammals.The fucosylation plays a key role in regulating important physiological functions via the modification of functional proteins.In the present study,mature 3-83 B cells were used to establish GDP-fucose transporter(slc35c1)knockout cell lines using CRISPR/Cas9.Two pGS-U6-gRNA vectors which included of gRNA sequences specific for Slc35c1 gene were designed,and the most effective pGS-U6-gRNA-slc35c1 vector was selected by AAL blot.The 3-83 cells were transfected with the obtained pGS-U6-gRNA-slc35c1.After selection of 400?g/ml G418 for about two months,the monoclonal cells were picked up and expanded.A stable slc35c1 gene knockout cell line,slc35c1-3-83-KO,was established and the silencing effects were detected by Western blot.To further establish the slc35c1 transgenic cell lines,we designed a point mutant of slc35c1 cDNA,and then slc35c1 cDNA was inserted to another viral vector(pLHCX).The pLHCX-Fut8-mutant vector was transformed into slc35c1-3-83-KO cells by safest reagent.After 200?g/ml the hygromycin screening for about a month,the re-expression of slc35c1 was detected by AAL blot.The slc35c1 gene recovery cells were referred to as slc35c1-3-83-KO-Re cells.To explore the function of fucosylation,we compared to the levels of cell growth and cell adhesion activity using 3-83,slc35c1-3-83-KO and slc35c1-3-83-KO-Re cells by MTT and cell adhesion assay and so on.Compare to the 3-83 cells,the fucosylation levels,cell growth and cell adhesion activity were significantly inhibited in slc35c1-3-83-KO cells,while those were restored in slc35c1-3-83-KO-Re cells,showing that fucosylation play an important role in the cell growth and cell adhesion of mature B-cell.