Rapid BAC selection for<i>tol2</i>-mediated transgenesis in zebrafish

Bussmann, Jeroen; Schulte‐Merker, Stefan

doi:10.1242/dev.068080

Cited by 171 publications

(174 citation statements)

References 29 publications

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“…However, recent reports have shown that much larger transgenes can be transposed. Tol2 vectors have been used to mobilize a bacterial artificial chromosomes (BAC) into the genome of early vertebrate embryos (42,43). Similarly, a piggyBac vector has been used to integrate sequences of up to 100 kb in length (44).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…BAC clone (number 74B2) from the DanioKey zebrafish BAC library containing the six2a and six3a genes was purchased from Source BioScience. Recombiant BACs were generated as previously described (36,37). For further details, see SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Gómez-Marín

Tena

Acemel

et al. 2015

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

154

147

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Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genomewide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.CTCF | TAD | Six cluster | regulatory landscapes | evolution

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“…To facilitate further analysis of abcc6a expression, a reporter construct was prepared by introducing the GAL4 element into the start codon of the abcc6a gene via BAC recombineering (Bussmann and Schulte-Merker, 2011). The mosaic expression of Tg(abcc6a: gal4;uas:gfp) at 7 dpf revealed GFP in the notochord and in cells near the operculum and cleithrum ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI

2015

Self Cite

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The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6.

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Rapid BAC selection fortol2-mediated transgenesis in zebrafish

Cited by 171 publications

References 29 publications

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

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

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI

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