Pig transgenesis by Sleeping Beauty DNA transposition

Jakobsen, Jannik E.; Li, Juan; Kragh, P. M.; Moldt, Brian; Lin, Lin; Liu, Ying; Schmidt, M.; Winther, K.D.; Schyth, Brian Dall; Holm, Ida E.; Vajta, Gábor; Bolund, Lars; Callesen, Henrik; Jørgensen, Arne Lund; Nielsen, Anders Lade; Mikkelsen, Jacob Giehm

doi:10.1007/s11248-010-9438-x

Cited by 58 publications

(60 citation statements)

References 56 publications

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“…As an alternative to the direct embryo injection described here, both the SB [50][51][52] and the piggyBac 53 transposon systems have been used to genetically modify porcine cells in vitro, which were subsequently used as donors in SCNT for pig transgenesis.…”

Section: Limitationsmentioning

confidence: 99%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

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2The pig has emerged as an important large animal model in biomedical and pharmaceutical research.We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer, and offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing as well as the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in one day; generation of germline-transgenic lines by using this protocol takes approximately one year.

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Section: Limitationsmentioning

confidence: 99%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

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

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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a b s t r a c tAlthough transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/ 119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%,11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

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“…All three of these transposable elements are functional in a wide range of host species, including chickens (22,23), and have been used to modify the germ line of many species (mouse, reviewed in ref. 13 [28][29][30]. Genome-wide mutational screens using gene-trap versions of these vectors have also been carried out successfully in several vertebrate species (20,(31)(32)(33)(34).…”

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

Proc. Natl. Acad. Sci. U.S.A.

155

127

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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...

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Pig transgenesis by Sleeping Beauty DNA transposition

Cited by 58 publications

References 56 publications

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

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

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

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