Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration
April DeLaurierB. Frank EamesBernardo Blanco‐SánchezGang PengXinjun HeMary E. SwartzBonnie UllmannMonte WesterfieldCharles B. Kimmel
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Abstract:
We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into one cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton.Keywords:
Otic vesicle
Transgenesis
Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.
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Minigene
Gene targeting
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The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.
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Transgenesis
Pronucleus
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Birds are useful research model for its easiness on various experimental treatments and subsequent analyses in ovo. Conventional transgenesis in this species has been achieved by direct infusion of retroviral vector into blastodermal cells of fleshly laid egg; however, this causes mosaic integration. A germ-cell based method using primordial germ cells (PGCs) is effective for transgenesis in birds, as they migrate to the sex cord via circulation and enable a germline modification. We recently produced transgenic quails by using lentiviral vector carrying enhanced green fluorescent protein (eGFP), and both conventional and PGC-mediated methods were possible for inducing stable transgenesis. We hypothesized that the expression of foreign gene can be affected by the characteristics of host genome neighboring transgene. In this study, we therefore mapped the location of transgene by gene walking and fluorescence in situ hybridization (FISH), and correlated its location and its expression. Five events of single copy integration in its respective genome, i.e., chromosome 5, 4, Z and 3 (both sense and antisense) were selected, and its expression was respectively analyzed by fluorescent imaging of whole body and frozen sections of transgenic quail. As results, the highest expression was noted in muscles in skeletal and digestive organs, regardless of transgenesis method. Among five integration events, transgene in chromosome 3 integrated in an antisense direction resulted the highest expression in all tissues. We showed that the extent of transgene expression is largely dependent on its location, and our findings can provide a valuable resource for devising key regulatory elements which enables constitutive transgene expression in bird.
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Transduction (biophysics)
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Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.
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Loss function
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Transgenesis
Cre-Lox recombination
Gene targeting
Pronucleus
Cre recombinase
Site-specific recombination
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Transgenesis
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Otic vesicle
FGF8
Lateral line
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