SRA

The vast diversity of mammalian adaptive antigen receptors allows for robust and efficient immune responses against a wide number of pathogens. The antigen receptor repertoire is built during the recombination and hypermutation of B and T cell receptor (B-, TCR) loci. V(D)J recombination rearranges these antigen receptor loci which are organized as an array of separate V, (D), and J gene segments. Which gene segments are recombined, and thus which particular antigen receptors are present in the host repertoire, depends on the three-dimensional architecture of the recombining locus. The endogenous retrovirus (ERV) mouse mammary tumor provirus (Mtv8) resides on mouse chromosome 6 interposed within the large array of light chain kappa (IgK) V gene segments. It was unknown, however, whether Mtv8 influences the BCR repertoire, as ERVs can contribute to changes in genomic architecture by driving high levels of local transcription. We generated Mtv8-deficient mice to determine if the ERV influenced V(D)J recombination and found no significant difference in the peripheral BCR repertoire of Mtv8-deficient mice compared to wildtype controls. Thus, Mtv8, the endogenous retrovirus in the Igk locus, does not influence BCR repertoires. Overall design: To investigate the role of Mtv8 in shaping the murine Ig repertoire, we used CRISPR/Cas9 to generate Mtv8-deficient C57BL/6J mice (KO). From WT and KO mice, we isolated splenic CD19+ cells using MACS and isolated RNA using guanidine thiocyanate extraction and CsCl gradient centrifugation. We then generated immunoglobulin libraries using the NEBNext Immune Sequencing Kit, specifically enriching for B cell receptor chains during the first PCR step. Libraries were sequenced by paired-end 300 bp sequencing on an Illumina MiSeq. Processing of data was performed using the pRESTO NEBNext Immune Sequencing Kit Workflow on Galaxy and subsequent analyses were performed in R.