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

Abstract

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...

Targeted gene knockout in chickens mediated by TALENs

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Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

“…Chicken PGCs are cultured and modified in vitro and then injected into embryos to generate transgenic chickens. Investigators have generated transgenic chickens and offspring efficiently using PB transposons (Macdonald et al, 2012;Park & Han, 2012). However, as is widely known, it is difficult to isolate many PGCs from embryos, and culturing PGCs in vitro is technically challenging and expensive.…”

“…The SB transposon system was the first used to try and increase the transgene efficiency in chicken and turkey cells (Kong et al, 2008), while a germ-line-competent chicken PGC line was developed with the PB transposon and transposase, and both of the efficiency of transgenesis and the expression level of transgene were improved with these modified PGCs (Park & Han, 2012). A transgenic chick can be generated with the PGCs modified in vitro using both PB and Tol2 transposons (Macdonald et al, 2012). Lu et al (2015) first demonstrated that a PB transposon mediated the insertion of a transgene into chicken embryos during developmental stages.…”

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

“…In the past few years, transposon-mediated transgenesis was efficient for production of genetically modified livestock, namely swine [15][16][17] and chickens [18].…”