b4galt1 grx1 rogfp1 ie gene sequence  (Addgene inc)

Journal: bioRxiv

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

doi: 10.1101/2022.11.16.516725

Figure Lengend Snippet: ( A ) MDCK and hCK cells were modified with recombinant lentiviruses containing transfer plasmids for the insertion of the B3GNT2 and/or B4GALT1 gene and the Hygromycin B resistance gene. The knock-in cells were selected with 300 µg/ml Hygromycin B. ( B ) RT-qPCR was performed with primers that anneal to both the human and dog B3GNT2, B4GALT1 , or ST6GAL1 genes. The values relative to the dog GAPDH gene were used, which were then normalized to the highest value of each gene. Mean and SD (n=3) are shown. ( C ) Mass spectrometry of the B3GNT2, B4GALT1, and ST6GAL1 proteins. Only peptides unique to human proteins were selected. All samples were normalized against tubulin beta and then normalized to the highest value of each protein. Mean and SD (n=3) are shown.

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer Lippincott-Schwartz, Addgene plasmid # 11929) [ ].

Techniques: Modification, Recombinant, Knock-In, Quantitative RT-PCR, Mass Spectrometry

Journal: bioRxiv

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

doi: 10.1101/2022.11.16.516725

Figure Lengend Snippet: The N -glycans from WT and B3GNT2/B4GALT1 knock-in MDCK and hCK cells were measured using mass spectrometry. ( A ) The relative abundance of high-mannose glycans was calculated as a percentage of all detected N -glycans (see Table S1-8). ( B ) The relative abundance of glycans (30-100%) with at least one SIA was calculated as a percentage of the total abundance of glycans with at least one LacNAc repeating unit (the glycans shown in ).

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer Lippincott-Schwartz, Addgene plasmid # 11929) [ ].

Techniques: Knock-In, Mass Spectrometry

Journal: bioRxiv

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

doi: 10.1101/2022.11.16.516725

Figure Lengend Snippet: The N -glycans from WT and B3GNT2/B4GALT1 knock-in MDCK and hCK cells were measured using mass spectrometry. ( A ) Chromatograms of hCK WT and hCK-B3GNT2 cells were constructed for the glycans with at least two and at most seven LacNAc repeating units. The extracted-ion-counts for the ten most abundant glycan features per LacNAc repeating unit group were summed to yield a chromatogram. ( B ) The N -glycans with at least one LacNAc repeating unit were analyzed for the number of LacNAc repeating units present and the relative abundance was calculated. Further analysis is presented in . Full glycan feature lists for each cell line are presented in Table S1-8.

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer Lippincott-Schwartz, Addgene plasmid # 11929) [ ].

Techniques: Knock-In, Mass Spectrometry, Construct, Glycoproteomics