Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production

Hiroaki Matsushita; Akiko Sano; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J Sullivan; Zhongde Wang; Yoshimi Kuroiwa

doi:10.1371/journal.pone.0090383

Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production

PLoS One. 2014 Mar 6;9(3):e90383. doi: 10.1371/journal.pone.0090383. eCollection 2014.

Authors

Hiroaki Matsushita¹, Akiko Sano², Hua Wu¹, Jin-An Jiao¹, Poothappillai Kasinathan³, Eddie J Sullivan¹, Zhongde Wang⁴, Yoshimi Kuroiwa²

Affiliations

¹ Sanford Applied Biosciences L.L.C., Sioux Falls, South Dakota, United States of America; Hematech, Inc., Sioux Falls, South Dakota, United States of America.
² Kyowa Hakko Kirin, Co., Ltd., Chiyoda-ku, Tokyo, Japan; Hematech, Inc., Sioux Falls, South Dakota, United States of America.
³ Trans Ova Genetics, Sioux Center, Iowa, United States of America; Hematech, Inc., Sioux Falls, South Dakota, United States of America.
⁴ Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America; Hematech, Inc., Sioux Falls, South Dakota, United States of America.

Abstract

Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Animals, Newborn
Antibodies, Monoclonal / immunology*
B-Lymphocytes / immunology
B-Lymphocytes / metabolism
Base Sequence
CHO Cells
Cattle
Cell Line
Chromosomes, Artificial, Human / genetics*
Cricetinae
Cricetulus
Female
Flow Cytometry
Gene Knockout Techniques*
Humans
Immunoglobulin G / genetics
Immunoglobulin G / immunology
Immunoglobulin Heavy Chains / genetics
Immunoglobulin Heavy Chains / immunology
Immunoglobulin kappa-Chains / genetics
Immunoglobulin kappa-Chains / immunology
Immunoglobulin lambda-Chains / genetics
Immunoglobulin lambda-Chains / immunology
Immunoglobulin mu-Chains / genetics
Immunoglobulin mu-Chains / immunology
Immunoglobulins / genetics*
Immunoglobulins / immunology*
Male
Molecular Sequence Data
Reverse Transcriptase Polymerase Chain Reaction
Sequence Homology, Nucleic Acid

Substances

Antibodies, Monoclonal
Immunoglobulin G
Immunoglobulin Heavy Chains
Immunoglobulin kappa-Chains
Immunoglobulin lambda-Chains
Immunoglobulin mu-Chains
Immunoglobulins

Grants and funding

This work was financed by internal funding of Hematech, Inc., which no longer exists and no longer has financial interest in the work. Hematech has transferred all of its business to Sanford Applied Biosciences, LLC. No current external funding sources for this study. Financial interest in this work is currently owned by Sanford Applied Biosciences, who did have a role in the decision to publish as well as the preparation of the manuscript. While it is considered beneficial to the interests of Sanford Applied Biosciences to publish this work, this consideration played no role in the study design, data collection and analysis and preparation of the manuscript. This is evidenced by the inclusion of now independent authors with no affiliation to Sanford Applied Biosciences.