Journal: BMC Biotechnology
Article Title: Enhanced transduction of colonic cell lines in vitro and the inflamed colon in mice by viral vectors, derived from adeno-associated virus serotype 2, using virus-microbead conjugates bearing lectin
Figure Lengend Snippet: Effect of the co-attachment of lectins to the microbead surfaces on the infectivity of AAV2-microbead conjugates . AAV2.CMV-LacZ was biotinylated with sulfo-NHS-LC-biotin at 50 μg/ml, followed by the removal of non-virion-associated biotinylation reagent by dialysis. AAV2-microbead conjugates were prepared by the attachment of biotinylated AAV2 particles to the surfaces of avidin-coated fluorescent microbeads (480 nm in diameter) (9.2 AAV2 particles per microbead). To these AAV2-microbead conjugates, a biotinylated form of each lectin was added in excess (0.2 μg biotinylated lectin per 10 7 avidin-coated microbeads), followed by the removal of unbound lectin molecules by centrifugation. The infectivity of these AAV2-microbead conjugates with and without lectin was analyzed on HeLa, COLO 205, and MIP-101 cell lines. Cells were cultured in 24-well plates at 37°C for 24 hr (initial cell number per well: HeLa, 5 × 10 4 ; COLO 205, 1 × 10 5 ; MIP-101, 7.5 × 10 4 ). AAV2-microbead conjugates bearing each lectin, along with free unmodified AAV2.CMV-LacZ, free biotinylated AAV2.CMV-LacZ, and AAV2-microbead conjugates without lectin, were applied to target cells (a total of 1 × 10 7 AAV2 particles per well) and incubated at 37°C for 48 hr. Cells were fixed with glutaraldehyde and stained for β-galactosidase activity using X-gal as the substrate. Then, the number of infected cells in each well was counted under a light microscope. Each datum shown is the average number of infected cells per well with a standard deviation (n = 16). A, free, unmodified AAV2.CMV-LacZ; B, free, biotinylated AAV2.CMV-LacZ; C, AAV2-microbead conjugates without lectin; D – J, AAV2-microbead conjugates bearing lectin (D, Con A; E, horse gram agglutinin; F, peanut agglutinin; G, castor bean agglutinin I; H, soybean agglutinin; I, furze gorse agglutinin I; and J, wheat germ agglutinin).
Article Snippet: The following lectins in biotinylated form were obtained from Vector Laboratories [the carbohydrate structure(s), to which each lectin binds, is indicated in bracket]: Con A from Jack bean (Canavalia ensiformis ) seeds [mannose]; agglutinin from horse gram (Dolichos biflorus ) seeds [N -acetylgalactosamine]; agglutinin from peanuts (Arachis hypogaea ) [galactosyl (β-1,3)N -acetylgalactosamine]; agglutinin I from castor bean (Ricinus communis ) seeds [galactose and N -acetylglucosamine]; agglutinin from soybean (Glycine max ) seeds [N -acetylgalactosamine and galactose]; agglutinin I from furze gorse (Ulex europaeus ) seeds [fucose]; and agglutinin from wheat germ (Triticum vulgaris ) [N -acetylglucosamine].
Techniques: Infection, Avidin-Biotin Assay, Centrifugation, Cell Culture, Incubation, Staining, Activity Assay, Light Microscopy, Standard Deviation