Targeted disruption of the porcine immunoglobulin kappa light chain locus

Abstract

Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (κ) light chain constant (Cκ) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted Cκ allele (heterozygous knockout or ±) were bred together to generate Cκ homozygous knockout (-/-) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from Cκ -/- pigs were devoid of κ-containing Igs. Furthermore, there was an increase in lambda (λ) light chain expression when compared to that of wild-type littermates (Cκ +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.

Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting

“…Animals with disrupted IgM genes have been proven to be B cell-and antibody-deficient Tesson et al, 2011). Unlike other large farm animals, such as sheep, goats, and cattles, pigs have only one Ig heavy chain locus that needs to be inactivated for complete Ig replacement (Kuroiwa et al, 2009;Ramsoondar et al, 2011). Our data proved that the frameshift mutation in the J H region of the IgM heavy chain created by the CRISPR/Cas9 system could disrupt IgM protein expression ( Fig.…”

“…J-region gene segment of the Ig heavy chain locus knockout/B cell-deficient pigs and Ig kappa chain-knockout pigs have been reported Ramsoondar et al, 2011). However, these studies were based on traditional gene targeting technology, M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 whose efficiency is notoriously poor due to the extremely low frequency of homologous recombination in somatic cells.…”

Antibody Repertoire Development in Fetal and Neonatal Piglets. XX. B Cell Lymphogenesis Is Absent in the Ileal Peyer’s Patches, Their Repertoire Development Is Antigen Dependent, and They Are Not Required for B Cell Maintenance

“…Addressing the role of the IPP has implications beyond comparative and veterinary immunology because swine are used as models for human disease in cases where mouse models fail (50)(51)(52), in studies on xenotransplantation (53), as a model for immuno-ontogeny (41), and in the generation of B cell knockout pigs as the first step toward preparing humanized Abs in a large xenogeneic species (54,55).…”

Emerging Alternative Production Systems

“…In addition, the endogenous bovine immunoglobulin μ-chain locus and the bovine prion protein have been knocked out [284,285], leading to an increase in the ratio of human antibodies as well as providing a source for the reduction of BSE risk during production of the biopharmaceutical. Apart from cattle, several other livestock such as pigs or rabbits are also being exploited now as a source for the generation and production of recombinant antibodies by deleting their endogenous immunoglobulin loci [286][287][288].…”