Basic Fibroblast Growth Factor Activates MEK/ERK Cell Signaling Pathway and Stimulates the Proliferation of Chicken Primordial Germ Cells

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151

138

Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications.genetic modification | programmable genome editing | germ-line chimera | poultry | egg protein

Section: Discussionmentioning

confidence: 89%

Targeted gene knockout in chickens mediated by TALENs

Park

Lee

Kim

et al. 2014

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151

138

“…PGCs from 6-d-old gonads of each White Leghorn (WL) embryo were prepared and maintained without loss of germ-cell integrity. In our previous report (2), chicken PGCs were maintained without any feeder layer, but in the present study chicken PGCs were subcultured onto mitomycin-inactivated mouse embryonic fibroblasts (MEFs) in 5-to 6-d intervals. Compared with the feeder-free procedure, the majority of chicken PGCs on MEFs grew as a single cell and could be subpassaged by gentle pipetting without any enzyme treatment.…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, an in vitro long-term culture system for stable PGC lines remains to be established in mammals, including the mouse. Only chicken PGCs have been propagated continuously in vitro without loss of germ-cell properties (1)(2)(3). Germ-line-competent PGC lines could be versatile tools for studying germ-cell biology, including the transcriptional regulation of microRNAs (4), and for creating transgenic chickens (1).…”

mentioning

confidence: 99%

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

Park

Han

2012

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153

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

“…Breeding from the resulting germline chimeras demonstrated that the cultured PGCs formed functional gametes by production of offspring derived from these cells. Germ-line transmission of cultured PGCs has now been demonstrated for three different chicken breeds (5)(6)(7).…”

mentioning

confidence: 99%

Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons

Macdonald

Taylor

Sherman

et al. 2012

155

127

The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro-and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.poultry | transgenesis | transposition P rimordial germ cells (PGCs) are specified in the early embryo and are the lineage-restricted stem cells for the germ cell population. In the chicken, segregation of PGCs from somatic cells occurs during the early stages of blastoderm formation (1-3). These cells accumulate in the germinal crescent region anterior to the forming head at day 1 of incubation [stage 4 Hamburger and Hamilton (HH)] and subsequently migrate via the circulatory system to the forming gonads on day 3 of incubation (stage 15 HH) (4). Chicken PGCs can be isolated from embryonic blood at this developmental stage and propagated indefinitely in culture (5). When returned to the circulatory system of an equivalent stage host embryo, the cultured PGCs migrated to and populated the forming embryonic gonad. Breeding from the resulting germline chimeras demonstrated that the cultured PGCs formed functional gametes by production of offspring derived from these cells. Germ-line transmission of cultured PGCs has now been demonstrated for three different chicken breeds (5-7).This in vitro system for the long-term propagation of chicken PGCs is the basis of a unique stem cell model for both the investigation of germ cell differentiation and the generation of genetically modified chickens. Thus far, chicken PGCs have proved highly resistant to genetic modification. Stable transfection of PGCs has been achieved by electroporation of plasmid DNA, at a frequency of ∼1 in 10 6 , but expression from integrated reporter constructs depended on t...