Transposon-mediated gene trapping in zebrafish

Kotani, Tomoya; Nagayoshi, Saori; Urasaki, Akihiro; Kawakami, Koichi

doi:10.1016/j.ymeth.2005.12.006

Cited by 67 publications

(67 citation statements)

References 17 publications

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“…This initial demonstration, coupled with the germ-line transmission of transposon-modified chicken PGCs, validates the prospect of conducting a large-scale mutational screen of gene function in chickens. Large-scale mutational gene-trap screens have been carried out in many species (20,31,32). Such an analysis in chickens would be a valuable and alternative model to the mutational screens being carried out in the mouse.…”

Section: Discussionmentioning

confidence: 99%

“…Tol2 integration sites were mapped by inverse PCR using the nested primers and conditions of ref. 20, with the following modifications: 1.0 μg of DNA was digested overnight with AluI, HaeIII, or PsiI to map 5′-junction fragments or with HaeIII and HindIII to map 3′-junction fragments. Digested DNA was heat treated and ligated overnight at 15°C, using T4 DNA ligase.…”

Section: Dna Transposon Vectorsmentioning

confidence: 99%

“…27; and pig,. 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).The aim of this study was to evaluate whether DNA transposons can be used to increase the efficiency of the genetic The authors declare no conflict of interest. …”

mentioning

confidence: 99%

“…The piggyBac and Sleeping Beauty transposons have been shown to integrate preferentially into transcriptional units in several cell types (17)(18)(19). Similarly, the Tol2 transposon frequently integrates into intragenic regions (20,21). 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.…”

mentioning

confidence: 99%

“…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%

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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.

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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%

Section: Dna Transposon Vectorsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…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%

See 3 more Smart Citations

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.

Self Cite

155

127

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Real‐time imaging of actin filaments in the zebrafish oocyte and embryo

et al. 2015

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Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis.

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Olfactory placode generates a diverse population of neurons expressing GnRH, somatostatin mRNA, neuropeptide Y, or calbindin in the chick forebrain

Murakami

Ohki‐Hamazaki

Uchiyama

2022

J of Comparative Neurology

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The olfactory placode (OP) of vertebrates generates several classes of migrating cells, including hypothalamic gonadotropin-releasing hormone (GnRH)-producing neurons, which play essential roles in the reproduction system. Previous studies using OP cell labeling have demonstrated that OP-derived non-GnRH cells enter the developing forebrain; however, their final fates and phenotypes are less well understood. In chick embryos, a subpopulation of migratory cells from the OP that is distinct from GnRH neurons transiently expresses somatostatin (SS). We postulated that these cells are destined to develop into brain neurons. In this study, we examined the expression pattern of SS mRNA in the olfactory-forebrain region during development, as well as the destination of OP-derived migratory cells, including SS mRNA-expressing cells. Utilizing the Tol2 genomic integration system to induce long-term fluorescent protein expression in OP cells, we found that OP-derived migratory cells labeled at embryonic day (E) 3 resided in the olfactory nerve and medial forebrain at E17-19. A subpopulation of green fluorescent protein (GFP)-labeled GnRH neurons that remained in the olfactory nerve was considered to comprise terminal nerve neurons. In the forebrain, GFP-labeled cells showed a distribution pattern similar to that of GnRH neurons. A large proportion of GFP-labeled cells expressed the mature neuronal marker NeuN.Among the GFP-labeled cells, the percentage of GnRH neurons was low, while the remaining GnRH-negative neurons either expressed SS mRNA, neuropeptide Y, or calbindin D-28k or did not express any of them. These results indicate that a diverse population of OP-derived neuronal cells, other than GnRH neurons, integrates into the chick medial forebrain.

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Transposon-mediated gene trapping in zebrafish

Cited by 67 publications

References 17 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

Real‐time imaging of actin filaments in the zebrafish oocyte and embryo

Olfactory placode generates a diverse population of neurons expressing GnRH, somatostatin mRNA, neuropeptide Y, or calbindin in the chick forebrain

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