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GFP expression in different tissues of Blk5, a mosaic transgenic mouse (FO). Bright field images of tissues are shown in the left column, and the corresponding fluorescent image is presented in the right column. (A) Whole brain as seen from above, looking at the dorsal side of the brain with the caudal end at the bottom of the image. (B) Testes. (C) Muscle tissue of the inner side of the left leg.
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Zebra finches are widely used for studying the basic biology of vocal learning. The inability to introduce genetic modifications in these animals has substantially limited studies on the molecular biology of this behavior, however. We used an HIV-based lentivirus to produce germline transgenic zebra finches. The lentivirus encoded the GFP regulated...
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... externally, also lacked GFP expression inter- nally. In Blk16, external examination showed GFP-expressing cells in eye tissue, but internal examination revealed GFP expression in the brain but not in other tissues. Blk5 had a similar external pattern of GFP expression as Blk16, along with expres- sion in the brain, muscles, and testicular tissue (Fig. ...
Citations
... We took advantage of a line of transgenic zebra finches that express green fluorescent protein (GFP) under the ubiquitin C (UBC) promoter. 12 The UBC-GFP transgenic strain was produced to demonstrate the efficiency of lentivirus-based transgenesis in songbirds, but GFP expression was not characterized in detail. Here we show that GFP is strongly expressed in a lineage that includes both young and mature neurons and that its expression is sparse enough and bright enough to enable single-cell tracking in vivo. ...
... Here we evaluated UBC-GFP zebra finches, a previously developed transgenic zebra finch line, 12 for the study of neuron migration and provided an in-depth quantitative characterization of neuron migration in the postnatal zebra finch brain. We found that migratory cells are present throughout HVC and surrounding tissue at high density, disperse in all directions, and may use vasculature as migratory scaffolding. ...
... Transgenic UBC-GFP songbirds used in the study were previously created using lentiviral mediated trangenesis. 12 To identify the transgene insertion site we used whole genome sequencing followed by alignment to the zebra finch genome (bTaeGut2.pat.W.v2, https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_008822105.2/) and plasmid FUGW (Addgene plasmid # 14883; http://n2t. net/addgene:14883; RRID:Addgene_14883). Article ll OPEN ACCESS Genomic DNA was obtained from blood. ...
... The lentivirus system has also been introduced to generate transgenic zebra finches (Taeniopygia guttata), a representative animal model of vocal learning. After the introduction of a lentivirus containing a human ubiquitin-C promoter-driven GFP transgene into very early stage embryos, 13% (3/23) of founders were germline transgenics [38]. Similarly, a transgenic zebra finch model in which cAMP response element binding protein (CREB) is regulated or expressed in the human mutant huntingtin (mHTT) gene was also developed using the lentiviral system to monitor vocal learning behavior and to model Huntington's disease [39,40]. ...
... The Forkhead box protein 2 (FOXP2) mutations in humans lead to developmental verbal dyspraxia (DVD) and lentivirus mediated FOXP2 gene knockdown in zebra finch results in abnormal speech production [198,199]. Transgenic zebra finches carrying the GFP gene under the control of the human ubiquitin-C promoter were generated and GFP-expressing cells located in the forebrain could be traced and analyzed [38,200]. Gonadal PGCs of zebra finch are heterogenous, and the signaling pathways contributing to their development differ from those of chickens [201,202]. ...
Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. However, in birds, the introduction of the CRISPR system into primordial germ cells (PGCs), a germline-competent stem cell, is considered a much more reliable approach for the development of genome-edited models. After genome editing, PGCs are transplanted into the embryo to establish germline chimera, which are crossed to produce genome-edited birds. In addition, various methods, including delivery by liposomal and viral vectors, have been employed for gene editing in vivo. Genome-edited birds have wide applications in bio-pharmaceutical production and as models for disease resistance and biological research. In conclusion, the application of the CRISPR system to avian PGCs is an efficient approach for the production of genome-edited birds and transgenic avian models.
... To date, the hatching success rate after manipulation in zebra finch ranges from 10 to 45%, and the germline transmission efficiency of the offspring has varied in the range of 4-22%, leaving some room for improvement. The low hatching rates and difficulties associated with embryo manipulation and fostering of hatched finches have hampered the effective and stable production of germline chimeric zebra finches [11][12][13][14][15]. ...
... The zebra finch is an important model animal for neurobiological research, and demand for the production of transgenic zebra finch is increasing continuously [6]. A small number of transgenic zebra finches with low germline transmission efficiency have been reported to date; however, there is a need to develop improved systems [12][13][14][15]. Here, we developed a method to increase the germline chimera production efficiency with minimal animal sacrifice, via reversible intratesticular transplantation of PGCs and busulfan treatment of recipient zebra finches. ...
... testicular germ cells had been eliminated by busulfan treatment, suggesting that the system established here improves the efficiency of germline chimera production in zebra finches (Supplementary Table S1). In a previous report describing a method of targeting PGCs in the zebra finch blastoderm by injecting a lentivirus, the hatching rate was 10-13%, 5-13% of the founders were germline chimeras, and the germline transmission efficiency of the offspring was about 6-22% [13][14][15]. Another study that injected in vitro-modified PGCs into the blastoderm reported that all hatched founders (hatching rate of 45%) were confirmed as germline chimeras, and the germline transmission efficiency of the offspring was about 4-22% [12]. ...
Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.
... Firstly, we checked the efficiency of their transduction with lentivirus containing GFP reporter in the presence of 10 and 50% of virus supernatant. These types of viral vectors are widely used for gene delivery to mammalian [32,33] and avian [34][35][36] cells. To produce transgenic ...
... Firstly, we checked the efficiency of their transduction with lentivirus containing GFP reporter in the presence of 10 and 50% of virus supernatant. These types of viral vectors are widely used for gene delivery to mammalian [32,33] and avian [34][35][36] cells. To produce transgenic birds, most of these studies injected the virus vectors into newly laid eggs. ...
The last decade was marked by a steep rise in avian studies at genomic and cellular levels. Cell lines are important tools for in vitro studies in cell biology and cytogenetics. We developed a simple method of primary somatic cell culture establishment from the ovaries of the great tits (Parus major) and testes of ten Passerine species, characterized the cellular composition of the ovary-derived lines using RT-PCR and immunolocalization of the tissue-specific markers and tested the efficiency of two methods of genetic transformation of the ovary-derived cell line. We found that the ovary-derived cell cultures of the great tit were composed of fibroblasts mainly, but also contained interstitial and granulosa cells. They were cultivated until the 10th passage without any noticeable decrease in their proliferative activity. The testis-derived cell cultures had lower proliferative potential. However, both ovary- and testis-derived cell cultures provided enough material for high quality mitotic metaphase chromosome preparations. The efficiency of its transduction with lentivirus containing a GFP reporter was very low, while electroporation with episomal vectors expressing GFP resulted in a high yield of GFP-positive cells. The proposed method could be used for the generation of high quality material for various cytogenetic and genomic studies.
... Germ line transgenesis of zebra finches have been achieved, but with low-efficiency (Agate et al., 2009;Abe et al., 2015;Liu et al., 2015). Recent reports have improved efficacy of transgenesis by direct lentiviral infection of cultured PGCs (Gessara et al., 2021), resulting in transgenic songbirds expressing eGFP in various tissues including neurons in the song circuits. ...
Rodents have been the dominant animal models in neurobiology and neurological disease research over the past 60 years. The prevalent use of rats and mice in neuroscience research has been driven by several key attributes including their organ physiology being more similar to humans, the availability of a broad variety of behavioral tests and genetic tools, and widely accessible reagents. However, despite the many advances in understanding neurobiology that have been achieved using rodent models, there remain key limitations in the questions that can be addressed in these and other mammalian models. In particular, in vivo imaging in mammals at the cell-resolution level remains technically difficult and demands large investments in time and cost. The simpler nervous systems of many non-mammalian models allow for precise mapping of circuits and even the whole brain with impressive subcellular resolution. The types of non-mammalian neuroscience models available spans vertebrates and non-vertebrates, so that an appropriate model for most cell biological questions in neurodegenerative disease likely exists. A push to diversify the models used in neuroscience research could help address current gaps in knowledge, complement existing rodent-based bodies of work, and bring new insight into our understanding of human disease. Moreover, there are inherent aspects of many non-mammalian models such as lifespan and tissue transparency that can make them specifically advantageous for neuroscience studies. Crispr/Cas9 gene editing and decreased cost of genome sequencing combined with advances in optical microscopy enhances the utility of new animal models to address specific questions. This review seeks to synthesize current knowledge of established and emerging non-mammalian model organisms with advances in cellular-resolution in vivo imaging techniques to suggest new approaches to understand neurodegeneration and neurobiological processes. We will summarize current tools and in vivo imaging approaches at the single cell scale that could help lead to increased consideration of non-mammalian models in neuroscience research.
... Also, to overcome the promoter competition in packaging cells and to improve the vector titer, hybrid ubiquitous promoters such as RSVp-5 LTR and CMVp-5 LTR were exploited [94]. Although the CMVp-5 LTR was successfully used in avian transgenesis ( Figure 3j) [83,95], hybrid RSVp-5 LTR efficiently improved the rate of germline chimera production [94]. The tissue-specific expression of eGFP controlled by a tissue-specific promoter in FUGW with hybrid CMVp-5 UTR successfully improved germline transmission in quail [83,96]. ...
... The tissue-specific expression of eGFP controlled by a tissue-specific promoter in FUGW with hybrid CMVp-5 UTR successfully improved germline transmission in quail [83,96]. Also, the utility of ubiquitin C promoter (UBCp) in FUGW was reported for the generation of transgenic finches with success [95]. No developmental silencing was observed in both instances [83,95]. ...
... Also, the utility of ubiquitin C promoter (UBCp) in FUGW was reported for the generation of transgenic finches with success [95]. No developmental silencing was observed in both instances [83,95]. Compared with highly efficient transgenesis in mice using the FUGW, transgenesis in quail and finch was not highly promising [92]. ...
Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-CRISPR and CRISPR era in the field of avian genome engineering.
... Zebra finch is an ideal organism for studying the basis of neurogenesis and speech disorders in humans, but transgenic studies are lacking, and the germline transmission efficiency is very low 1,2,7,8 . Generation of germline chimeras using germline-competent stem cells, such as PGCs and SSCs, is regarded as an efficient approach to produce transgenic animals of avian species. ...
Zebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.
... This manipulation boosted GFP expression 10-fold in the zebra finch cells, whereas the high fluorescence previously seen in chicken cultures was unaltered, supporting the author's inference that the lost gene regions were inhibiting functionality. This finding solves the puzzle of the low neuronal infectivity of VSV-G-pseudotyped lentiviruses when injected into zebra finch brain (10) and the relatively rare success in using them for songbird transgenics (11). ...
... [34][35][36] Our laboratory and others showed an improved germline transmission of transgenes in chicken, quail and zebra finch using lentiviral-based vectors. [36][37][38][39] Recently, adenoviruses were used to deliver a Cas9 transgene and a guide RNA directly to the quail blastoderm. 40,41 Initially, authors targeted the quail melanophilin gene, resulting in grey plumage of homozygous GE quail offspring produced from the founder birds, whereas heterozygous and wild type quail exhibited dark brown plumage. ...
Avian species are used as model systems in research and have contributed to ground-breaking concepts in developmental biology, immunology, genetics, virology, cancer and cell biology. The chicken in particular is an important research model and an agricultural animal as a major contributor to animal protein resources for the global population. The development of genome editing methods, including CRISPR/Cas9, to mediate germline engineering of the avian genome will have important applications in biomedical, agricultural and biotechnological activities. Notably, these precise genome editing tools have the potential to enhance avian health and productivity by identifying and validating beneficial genetic variants in bird populations. Here, we present a concise description of the existing methods and current applications of the genome editing tools in bird species, focused on chickens, with attention on animal use and welfare issues for each of the techniques presented.
... Songbirds are major animal models for studying the genetic and neural basis of vocal learning and communication (Mello, 2014;Mooney, 2020;Prather et al., 2017) as well as sex-hormone-dependent brain development (Balthazart et al., 2010;Gahr, 2007;McCarthy and Arnold, 2011) and adult neurogenesis (Goldman and Nottebohm, 1983;Paton and Nottebohm, 1984). For the zebra finch, transgenic models have been successfully developed (Abe et al., 2015;Agate et al., 2009;Liu et al., 2015). Agate et al. (2009) were the first to inject lentiviral vectors for GFP into the blastodisc of freshly laid zebra finch eggs in order to target the primordial germ cells (PGCs), the precursors of spermatocytes and oocytes. ...
... For the zebra finch, transgenic models have been successfully developed (Abe et al., 2015;Agate et al., 2009;Liu et al., 2015). Agate et al. (2009) were the first to inject lentiviral vectors for GFP into the blastodisc of freshly laid zebra finch eggs in order to target the primordial germ cells (PGCs), the precursors of spermatocytes and oocytes. However, due to the inefficiency of the method, only two other transgenic models have been generated over the past 10 years using this method (Abe et al., 2015;Liu et al., 2015). ...
... When grown in cell clumps after 7 DIV, zfPGCs exhibited high transduction rates using lentiviral vectors such that almost every zfPGC expressed the reporter gene. Previously, the transduction and transmission efficiency of lentiviral vectors after injection at the blastodermal stages was low (Agate et al., 2009). In particular, lentiviral vectors transduced zfPGCs much less efficiently in ovo compared with cultured zfPGCs growing in clumps. ...
The ability to genetically manipulate organisms has led to significant insights into functional genomics in many species. In birds, manipulation of the genome is hindered by the inaccessibility of the one-cell embryo. During embryonic development, avian primordial germ cells (PGCs) migrate through the bloodstream and reach the gonadal anlage, where they develop into mature germ cells. Here, we explored the use of PGCs to produce transgenic offspring in the zebra finch, which is a major animal model for sexual brain differentiation, vocal learning, and vocal communication. Zebra finch PGCs (zfPGCs) obtained from embryonic blood significantly proliferated when cultured in an optimized culture medium and conserved the expression of germ and stem cell markers. Transduction of cultured zfPGCs with lentiviral vectors was highly efficient, leading to strong expression of the enhanced green fluorescent protein. Transduced zfPGCs were injected into the host embryo and transgenic songbirds were successfully generated.
