3: Generation of TFG negative clones of the CH12.LX cell line. A, schematic illustration of the pX458 plasmid containing a U6 promoter driven single guide RNA (sgRNA), a chicken β-Actin promoter driven sequence composed of CRISPR-associated protein 9 (Cas9) and enhanced green fluorescent protein (eGFP) fused with a E2A self-cleaving peptide linker and a bacterial Ampicillin resistance (Amp R ). B, workflow of TFG KO cell generation: CH12.LX cells were electroporated with pX458_sgRNATFG followed by single cell subcloning and western blot analysis. C, representative western blot of screened CH12.LX CRISPR treated subclones. Clone IDs on top marking TFG KO cells (bold black), normal TFG expressing clones (black) and samples that were excluded due to low Actin expression (grey). Molecular weight standard is marked on the left. D, confirmation of CRISPR/Cas9 mediated mutations by Sanger sequencing of representative CH12 subclones (1C11, 1E10, 3G7) tested negative for TFG expression on western blot compared to the wildtype tfg sequence. Asterisks indicate sequence identity with WT, tfg start codon (green), PAM sequence (red) and Cas9 cut side are highlighted.

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... was unaltered. Moreover, fecal IgA was not influenced by the TFG genotype ( Fig. 2.28A). Characterization of isotype distribution within the PB/PC compartment showed that IgG ASC are most abundant in the spleen, IgM ASC predominately in the mLNs, while bone marrow and Peyer's patch plasmablasts and plasma cells expressed IgA in 75% of all ASCs (Fig. 2.23). Bone marrow PB/PC frequencies in TFG +/-mice were normal indicating an unaltered contribution of the bone marrow to IgA serum titers. Gut-associated plasma cells of the IgA isotype are described to primarily secrete into the gut lumen via pIg receptors. Thereby, formation of dimeric IgA via the joining chain (IgJ) is critical and ...