Date on Master's Thesis/Doctoral Dissertation

5-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Gregg, Ronald

Committee Co-Chair (if applicable)

Mitchell, Thomas C.

Committee Member

Mitchell, Thomas C.

Committee Member

Samuelson, David

Committee Member

Watson, Corey

Committee Member

Yan, Jun

Author's Keywords

genomic; germline diversity; mouse antibody; repertoire developement

Abstract

Given the diversity and complexity within immunoglobulin (IG) loci, effective mouse models first require characterization of intra-strain differences and construction of high-quality reference assemblies for IG loci in several representative strains. To understand light chain germline diversity across biomedically significant mouse strains, we profiled the expressed IGK and IGL repertoires of 18 commonly used laboratory mouse strains using AIRR-seq. Across strains, we observed germline IGKV sequences shared by three different IGK haplotypes and a more conserved IGLV germline repertoire among common laboratory strains. Pacific Biosciences (PacBio) Single-Molecule Real-Time (SMRT) sequencing was used to sequence and assemble bacterial artificial chromosomes (BAC) clones spanning the IGH locus in BALB/cByJ and the IGK locus in NOD/LtJ, which represented divergent IG haplotypes. We assembled the BALB/cByJ-IGH assembly into five independent contigs containing 192 functional and 135 non-functional IGHV genes, 30 IGHD genes, 4 IGHJ genes, and 8 IGHC genes. The NOD/ShiLtJ-IGK assembly was assembled into two independent contigs, which contained 82 functional and 31 non-functional IGKV genes. These data guided construction of congenic strains on a C57BL/6 background that carried divergent BALB/cByJ or NOD/ShiLtJ IGH or IGK loci from, respectively. In addition, bulk AIRR-seq data from the BALB/cByJ-IGH congenic strain showed that divergent IGH haplotype influenced usage frequencies of germline IGKV and IGLV repertoire. Overall, this work revealed significant unexplored IG haplotype diversity through AIRR-seq, generated new IG reference assemblies, identified incomplete germline gene databases that lacked haplotype diversity, and provided evidence that heavy and light chain pairing frequencies are likely influenced by underlying IG haplotype variation.

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