Creative Enzymes provides high-quality creatinase (EC 3.5.3.3), creatininase (EC 3.5.2.10), and sarcosine oxidase (EC 1.5.3.1), which are raw materials used in the production of creatinine assay kits. The viruses were removed by washing the cells three times with medium, and luciferase expression was monitored at 24 h posttransduction on a VICTOR2 luminometer (Perkin-Elmer, Wellesley, MA) using a firefly luciferase assay kit (Promega). The fluorescence intensity was detected using a ScanArray 5000 (Perkin-Elmer Inc.) confocal scanner. Binding of lectin to cells was detected using fluorescence microscopy. If you loved this article and you would like to get much more information about sialic acid manufacturers (navigate to these guys) kindly go to our own web page. The preincubation solution was removed, and medium containing 100 μg/ml lectin and 2 × 108 rAAV particles was added. Lectin staining of HepG2 cells, Pro-5 cells, and Cos-7 cells was performed by incubation with fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA), Maackia amurensis lectin (MAA), or Sambucus nigra lectin (SNA) (Vector Laboratories Inc.), as described in reference 44. Briefly, cells growing in 24-well plates (approximately 2 × 105 cells/well) were chilled to 4°C, and then lectin (10 μg/ml) was added to the respective media. Briefly, Cos-7 cells were plated at a density of 2 × 104 cells/well in a 48-well plate. The cells were then cultured with the aforementioned inhibitors for 24 h and transduced with rAAV.
The cells were transduced with a constant amount of either AAV1 or AAV6 vector expressing luciferase in the presence of a 200-fold excess of AAV1, AAV6, or AAV2 as competitor. As shown in Fig. Fig.3,3, AAV2 bound and transduced both the parental cell line (Pro-5) and the sialic acid-deficient mutant (Lec-2) with similar efficiencies. Transduction of cells by luciferase-expressing AAV1 or AAV6 vectors in the presence of competing λ phage DNA-containing AAV1, AAV6, or AAV2 vectors. To further investigate the role of α2,3 and α2,6 sialic acids and their linkage specificities in AAV1 and AAV6 transduction, we carried out resialylation on sialic acid-deficient Lec-2 cells using sialyltransferase and CMP-sialic acid. WGA, which binds to all linkage forms of sialic acid, blocked both AAV1 and AAV6 transduction on all the cells tested. To further test if sialic acid is needed for efficient transduction by AAV1 and AAV6, we measured binding to and transduction of CHO cells deficient in cell surface sialic acid.
N-acetylneuraminic acid sialic acid powder is found in a wide variety of substances and tissues in animals and humans, occurring most abundantly in glycoproteins and glycolipids. In humans, concentrations are found in the brain and kidney as well as many other tissues. The data from these assays were complemented by a glycan array analysis which determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc as well as glycoproteins containing NeuAcα2-3/α2-6Galβ1-4GlcNAc N-linked glycans. The AAV serotype helper plasmids pXR1, pXR2, pXR4, and pXR5, as well as the plasmid pXX6-80 containing the adenovirus helper genes for AAV replication, have been described elsewhere (32, 47). AAV6 helper plasmid pXR6 was kindly provided by Joseph Rabinowitz (Thomas Jefferson University). Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline. These cell lines are derived from different origins, including Pro-5, HepG2, Cos-7 (Fig. (Fig.2),2), and HeLa (data not shown) cells, suggesting that this observation is not unique to airway cells. There was a 12- or 98-fold decrease in AAV6 transduction following neuraminidase treatment on HepG2 and Pro5 cells, respectively, in contrast to a 5- or 37-fold decrease in AAV1 transduction on these two cell lines.
MAA blocked AAV1 and AAV6 transduction on Pro-5 and Cos-7 cells, which display α2,3 sialic acid on their surfaces, but not on HepG2 cells, which display α2,6 sialic acid. AAV1 and AAV6 are two closely related viruses, with only six amino acid differences in their capsid regions. A printed slide was incubated with AAV1 capsids (at 200 μg/ml), and then a capsid monoclonal antibody (generated in collaboration with Colin Parrish) was overlaid on the bound capsids followed by a FITC-labeled secondary antibody (at 5 μg/ml). The sialic acid that facilitates AAV1 and AAV6 transduction localizes on glycoproteins rather than on glycolipids. At the concentration of 1 mM, N-benzyl GalNac inhibited AAV4 transduction by 10-fold. In contrast, only marginal or no inhibition was seen for AAV1, AAV6, or AAV2 transduction, indicating that AAV1 and AAV6 do not use O-linked sialic acid for transduction. Cos-7 cells were treated with the indicated doses of N-benzyl GalNAc (A) or tunicamycin (B) for 24 h prior to transduction. Cell binding assay. Pro-5 cells were treated with neuraminidase from Vibrio cholerae or mock treated prior to adding AAV1-luc, AAV6-luc, and AAV2-luc at 4°C. Virus binding was determined by quantitative dot blot hybridization.