title: IgG variable region and VH CDR3 diversity in unimmunized mice analyzed by massively parallel sequencing.
abstract: Most antigen-specific mouse antibodies have been derived by hybridoma technology, predominantly through use of the Balb/c strain. Much of the Balb/c germline repertoire of variable genes (V regions) is known. However, there is little information about the background expressed repertoire of IgG antibodies in mice, which reflects the baseline against which antigen-specific antibodies are generated through immunization. To assess this baseline repertoire, RNA was isolated from splenic B-cells enriched for expression of IgG from three mice. The RNA was individually amplified with three distinct PCR primer sets for comprehensive recovery of the heavy and light chain variable regions. Each PCR product was independently subjected to deep sequencing using 454 pyro-sequencing technology and analysed for redundancy, open reading frame, germline representation, and CDR3 sequence of the heavy chain variable region (VH CDR3) within and across the primer sets and mice. A highly skewed abundance of heavy and light chain variable gene usage was observed for all three primers in all three mice. While showing considerable overlap, there were differences among these profiles indicative of primer bias and animal-to-animal variation. VH CDR3 sequences were likewise highly skewed indicating that the heavy chain genes profiles substantially reflected individual antibodies. This observation was confirmed through analysis of randomly selected complete heavy chain variable sequences. However, there was very little redundancy in VH CDR3 sequences across the different mice. We conclude that the background IgG repertoire in young, unimmunized mice is highly skewed within individual mice and is diverse among them, a pattern similar to that observed in highly immunized mice.
authors.affiliation: Biologics Research, 1400 McKean Road, Spring House, PA 19477, United States.
authors.forename: Jin
authors.initials: J
authors.lastname: Lu
authors.forename: Tadas
authors.initials: T
authors.lastname: Panavas
authors.forename: Kim
authors.initials: K
authors.lastname: Thys
authors.forename: Jeroen
authors.initials: J
authors.lastname: Aerssens
authors.forename: Michael
authors.initials: M
authors.lastname: Naso
authors.forename: Jamie
authors.initials: J
authors.lastname: Fisher
authors.forename: Michael
authors.initials: M
authors.lastname: Rycyzyn
authors.forename: Raymond W
authors.initials: RW
authors.lastname: Sweet
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001483:Base Sequence; D059014:High-Throughput Nucleotide Sequencing; D007114:Immunization; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007144:Immunoglobulin J-Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D017423:Sequence Analysis, RNA; D061905:Single-Domain Antibodies / Q000235:genetics*
chemical_list: D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007144:Immunoglobulin J-Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D061905:Single-Domain Antibodies
keywords: Massively parallel sequencing; Mouse IgG1 repertoire