![]() ![]() Tissue extraction and sequencing were performed as described previously. We now present these data on the splenic repertoire of conventionally housed, unimmunized, unchallenged, adult C57BL/6J mice. Knowing that there can be significant mouse-to-mouse variability in the Ig repertoire and to minimize the impact any one mouse might have in the validation data set, we chose to pool multiple mice specifically for these validation experiments. We also wanted some background data about the Ig repertoire in the normal B6 mouse population. During the development of methodology to assess Ig-gene usage by mice subjected to space flight we performed multiple HTS runs to validate sample preparation, bioinformatic methodology, and reproducibility. While these practices increase the likelihood of recovering rare Ig sequences and allow for the dissection of the antibody repertoire by B-cell populations, cell sorting may not be possible within the design of certain experiments. The antibody repertoire is traditionally assessed through the amplification of Ig sequences that have been isolated from sorted B cell populations. Due to the cost of these experiments, creating datasets that can be mined by our lab or others is important. More specifically, our lab is interested antibody repertoire dynamics within the context of spaceflight. Our long-term goals are to investigate the repertoire of B cells in mice in space and how it changes in response to antigen challenge. HTS has accelerated the characterization of the widely differing human Ig haplotypes, and strain-specific gene segment usage in mice. With the development of HTS, we are now able to detect the differences between or among B-cell repertoires such as B2 (adaptive antibodies) and B1 (natural antibodies) B cells or memory and naïve repertoires. Repertoires can serve as a fingerprint or snapshot of the current immune-system status and these types of data have been used to explore the development of host defense to infectious disease, cancer, autoimmune disease, and early disease detection. The antibody repertoire has been examined in many studies by high-throughput sequencing (HTS) and fully mapped in the zebrafish. Diversity of the antibody repertoire results from four main components: the initial germ line (inherited), diversity from recombination of that germline, the imprecisions during V(D)J recombination, and somatic mutations. The total collection of antibody specificities present within an individual is known as the antibody repertoire. Antibodies are further characterized by the constant region, or isotype, which is influenced by the stage of B-cell development and antigen specificity. Of the CDRs, CDR3 contributes the most to binding specificity. CDR3 consists of a combination of V-, (D-, heavy-chain), and J-gene segments. CDR1 and CDR2 are encoded in the V-gene segment. There are three complementarity determining regions (CDR). The heavy chain is formed from V-, D-, and J-gene segments combined with a constant region, while light chains lack a D-gene segment. ![]() Antibodies consist of heterodimers of heavy and light chains. Īs B cells develop, they rearrange Variable- (V), Diversity- (D), and Joining- (J) gene segments, which combine with a constant region to form the antibody structure. These cells express surface immunoglobulin (Ig) receptors and secrete these same proteins as antibodies into the serum after differentiation into plasma cells. ![]() This study presents a unique, non-primer biased glimpse of the conventionally housed, unimmunized antibody repertoire of the C57BL6/J mouse.ī cells are an important part of the adaptive immune system, arising from hematopoietic stem cell precursors. There also was some skewing in the V-gene segments that were detected depending on chromosomal location. Despite the similar usage of individual gene segments, the repertoire of individual B-cell CDR3 amino acid sequences in each mouse pool was highly varied, affirming the combinatorial diversity in the B-cell pool that has been previously demonstrated. Individual V-, D-, and J-gene segment usage was uniform among the three mouse pools, particularly in highly abundant gene segments, with low frequency V-gene segments not being detected in all pools. We recovered over 90,000 heavy-chain and over 135,000 light-chain immunoglobulin sequences. We studied the antibody repertoire from pooled, splenic tissue of unimmunized, adult female C57BL/6J mice, using high-throughput sequencing (HTS) without amplification of antibody transcripts. Antibodies are heterodimers of heavy- and light-chains encoded on separate loci. Antibody specificity and diversity are generated through the enzymatic splicing of genomic gene segments within each B cell. ![]()
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