Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells

Abstract

Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.

Keywords: B-cell development; avian immunology; genome editing.

Fig. 1.

JH segment knockout chickens were produced by gene targeting in primordial germ cells followed by germ-line transmission of injected PGCs. (A) Diagram of the WT IgH locus (top line) with its single functional VH gene, a subset of the D cluster (D cluster is not fully mapped, as indicated by the break), single JH gene, and Cμ constant region. Vector IgH KO2 replaces 390 bp surrounding the JH gene segment with a selectable marker cassette. The JH-KO allele is shown on the bottom line, and positions of primers and Southern blot probes are indicated. R, EcoRI restriction site. (B) Genotyping of cell lines and birds by PCR. Primers were specific for the WT allele (primers 1 and 2) and the JH-KO allele (primers 1 and 3). (C) Southern blot analysis was done on EcoRI-digested gDNA from transgenic birds. (D) Germ-line transmission rates of three of the JH-KO targeted cell lines in chimeric roosters are displayed. Results from six roosters are shown.

Fig. 2.

IgM and IgY expression are eliminated, and B-cell migration to the periphery is blocked in JH-KO/JH-KO birds. (A) Relative amounts of total plasma IgM and IgY from WT (square), JH-KO/+ (triangle), and JH-KO/JH-KO (diamond) chickens were compared by ELISA at day 35 after hatch. Mean and SD of five hens per group are shown. (B) FACS analysis was performed on peripheral blood lymphocytes (PBLs) and spleen of 5-wk-old chickens and the bursa of 3-d-old chickens. B cells were stained with anti–chicken-Bu-1 (AV20), anti-chicken-Cµ (M1), and anti–chicken-lambda (L-1). Gamma/delta T cells were stained with anti–chicken-TCRγδ (TCR1), α/β T cells with anti–chicken-TCRαβ/Vβ1 (TCR2), and anti–chicken-TCRαβ/Vβ2 (TCR3) and chicken thrombocytes with a cross-reactive anti–human-CD51/61 (23C6). Percentages are shown on the y axis. Mean and SD of four birds per group are shown. Significance was calculated by ANOVA followed by Tukey test. **P < 0.01.

Fig. 3.

JH-KO/JH-KO chickens show reduced weight of lymphoid organs. (A) Gain in body weight was monitored until day 54 after hatch. Mean and SD of five birds per group are shown. Body weights of birds at embryonic day 18 (B), day 28 (C), and day 54 (D) after hatch were measured. Ratio between spleen or bursa weight (mg) and bodyweight (g) was calculated. Mean and SD of three birds per group are shown. Significance was calculated by ANOVA followed by Tukey test. *P < 0.05; **P < 0.01.

Fig. 4.

Effect of the JH segment knockout on the morphology of the bursa of Fabricius. The bursa of Fabricius of WT, JH-KO/+, and JH-KO/JH-KO chickens was prepared at embryonic day 14 and 18 and 1 and 28 d after hatch. Tissue sections of three birds per group were prepared. H&E staining was performed. One representative picture of every development stage and genotype is shown.

Fig. 5.

Separation of B-cell follicles by the cortico-medullary junction. Sections from frozen samples of the bursa from birds at 28 d after hatch were prepared. The basement membrane forming the cortico-medullary junction was stained using a cross-reactive anti–human-desmin (D33) antibody. The antibody was detected using the Vector ABC Kit followed by the Vector DAB Kit. One representative picture per group and staining is shown.

Fig. 6.

Distribution of T and B cells in homozygous JH segment knockout birds. Sections from frozen samples of the bursa of Fabricius (A) and the spleen (B) from birds at 28 d after hatch were prepared. B cells were stained with an anti–chicken-Bu-1 (AV20) antibody. IgM was stained using anti–chicken-Cµ (M1). T cells were stained using an anti–chicken-CD3 (CT-3) antibody. All antibodies were detected by an anti–mouse-IgG Cy3. One representative picture per group and staining is shown.