piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

Abstract

Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line-competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications.

Fig. 1.

G418-selected SNUhp26 PGCs after transfection with a piggyBac CMV-GFP expression vector. (A) G418-selected SNUhp26 PGCs (p48) for 61 d. Flow-cytometry analysis represented that G418-selected PGCs constantly expressed GFP. Genomic PCR results indicated that SNUhp26 was a male-derived PGC line. (Scale bar, 50 μm.) (B) PCR analysis of G418-selected PGCs using GFP-specific primers and Neo^R-specific primers. (C) RT-PCR analysis of G418-selected PGCs for germ-cell–specific (vasa and dazl) and stem-cell–specific genes (PouV and nanog). SSEA1-positive intact PGCs from blood vessel of 53-h embryos or 6-d-old embryonic gonads expressed both germ-cell– and stem-cell–specific genes, whereas SSEA1-negative somatic cells did not. (D) Flow cytometry analysis of G418-selected PGCs with SSEA1 and EMA1, which are specific antibodies for chicken germ cells. Phycoerythrin (PE)-conjugated secondary antibody for mouse IgM was used and the results were analyzed by flow cytometry.

Fig. 2.

Detection of GFP-expressing PGCs in recipient embryonic gonads and GFP-expressing transgenic chickens. Detection of GFP-expressing donor PGCs in recipient testes of 18-d-old (A) and hatched chicks (B) by confocal laser scanning microscope. Fluorescent illustrations of hatched chicks (C, the left chick is transgenic and the right chick is a normal hybrid), and head (D), wing (E), and breast (F) of a 10-wk-old transgenic chicken. Transgenic chickens showed strong GFP expression regardless of age. (G) Genomic PCR analysis of transgenic offspring. Transgenic progenies (lanes 1, 3, 4, 7, and 8) were positive for both GFP primers and Neo^R primers. Nontransgenic hybrids from the test-cross were negative control chicks. (H) Identification of the piggyBac GFP transgene site by DNA walking. A transgene was integrated into the distal end of chromosome 20. No functional gene or transcript was found within ∼0.1 Mb of the transgene integration site. Alignment of the transgene-flanking sequences from DNA walking analysis was conducted using the BLAST Assembled Genome Database (http://blast.ncbi.nlm.nih.gov).