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-linecompetent 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.germ-line chimera | piggyBac transposon | avian

Targeted gene knockout in chickens mediated by TALENs

“…At this time, chicken PGCs can be successfully maintained in vitro without the loss of germ cell integrity (32,33), and generating transgenic chickens is feasible using transposon elements in cultured chicken PGCs (18,34). This study demonstrated the creation of specific knockout chickens using TALENs.…”

“…In a previous report (18), we established a male chicken PGC line (SNUhp26) from WL embryonic gonads at day 6 (stage 28). The SNUhp26 PGC line was maintained and subpassaged with knockout DMEM (Invitrogen) supplemented with 20% FBS (Invitrogen), 2% chicken serum (Sigma-Aldrich, St. Louis, MO), 1× nucleosides (Millipore, Temecula, CA), 2 mM L-glutamine, 1× nonessential amino acids, β-mercaptoethanol, 10 mM sodium pyruvate, 1× antibiotic-antimycotic (Invitrogen), and human basic fibroblast growth factor (10 ng/mL; Koma Biotech, Seoul, Korea).…”

“…No GFP expression was observed in either of the mutant PGCs or donor-derived chicks, indicating that the transgene was not randomly integrated into the chicken genome during transfection or sorting procedures. In our previous report, the CMV promoter-controlled GFP gene was strongly expressed in transgenic chicks as well as in PGCs that contained a single transgene copy in their genome (18).…”

“…Genetic engineering techniques, such as stem cell-based and somatic cell nuclear transfer-mediated modifications, are difficult to apply in avian species because of technical difficulties associated with manipulating avian oocytes and accessing the oocyte nucleus. To overcome the technical obstacles of genomic modification in chickens, we recently established chicken primordial germ cell (PGC) lines with germ-line transmission capacity (18). We applied the same combination of techniques to the PGC line, using fluorescence-positive separation 1 d after cotransfection with the GFP vector and OV TALEN 2 set.…”

Cultivation and Biological Characterization of Chicken Primordial Germ Cells

“…Chicken as an acceptance model organism (Cogburn et al 2007) have advantages of population distribution and samples can be obtained easily. PGCs have been obtained mainly from germinal crescent (Petitte et al 1990, Park andHan 2012) and embryonic blood (van de Lavoir et al 2006, Macdonald et al 2010, Miyahara et al 2014, Naito et al 2015. However, their numbers have been quite limited (Park et al 2003) and couldn't provide sufficient materials for the transgenic manipulation (Shiue et al 2009).…”