Highly Efficient Genome Modification of Cultured Primordial Germ Cells with Lentiviral Vectors to Generate Transgenic Songbirds

Gessara, Ivana; Dittrich, Falk; Hertel, Moritz; Hildebrand, Staffan; Pfeifer, Alexander; Frankl-Vilches, Carolina; McGrew, Mike; Gahr, Manfred

doi:10.1016/j.stemcr.2021.02.015

Cited by 15 publications

(10 citation statements)

References 47 publications

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“…Zebra finch is a vocal learning species within the clade Neoaves and has a fully sequenced genome; thus, it is used increasingly for neurobiological research. Accordingly, interest in zebra finch PGCs is increasing ( Warren et al, 2010 ; Mello, 2014 ; Jung et al, 2019 ; Gessara et al, 2021 ). Here, we used an scRNA-seq approach to characterize zebra finch PGCs.…”

Section: Discussionmentioning

confidence: 99%

“…Zebra finch PGCs have distinct migratory and numerical characteristics at early developmental stage from those of other avian species. And the zebra finch is used for basic research and germline transgenics, indicating the importance of further germ cell research ( Jung et al, 2019 ; Gessara et al, 2021 ). In zebra finch, PGCs are widely distributed in the early embryo, complete their migration to the germinal crescent at Hamburger Hamilton stage (HH) 6 and enter the circulation through the anterior vitelline vein at HH stage 10.…”

Section: Introductionmentioning

confidence: 99%

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Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata)

Jung

Seo

Kim

et al. 2021

Front. Cell Dev. Biol.

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Primordial germ cells (PGCs) are undifferentiated gametes with heterogeneity, an evolutionarily conserved characteristic across various organisms. Although dynamic selection at the level of early germ cell populations is an important biological feature linked to fertility, the heterogeneity of PGCs in avian species has not been characterized. In this study, we sought to evaluate PGC heterogeneity in zebra finch using a single-cell RNA sequencing (scRNA-seq) approach. Using scRNA-seq of embryonic gonadal cells from male and female zebra finches at Hamburger and Hamilton (HH) stage 28, we annotated nine cell types from 20 cell clusters. We found that PGCs previously considered a single population can be separated into three subtypes showing differences in apoptosis, proliferation, and other biological processes. The three PGC subtypes were specifically enriched for genes showing expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Additionally, we discovered a novel biomarker, SMC1B, for gonadal PGCs in zebra finch. The results provide the first evidence of substantial heterogeneity in PGCs previously considered a single population in birds. This discovery expands our understanding of PGCs to avian species, and provides a basis for further research.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata)

Jung

Seo

Kim

et al. 2021

Front. Cell Dev. Biol.

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“…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. CRISPR/Cas9 editing has been achieved in quail and chicken, and adenovirus-mediated CRISPR/Cas9 delivery has been used to edit zebra finch PGCs, but no adult mutant has yet been generated in zebra finch ( Lee et al, 2019 ; Xu et al, 2020 ; Jung et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

A Model of Discovery: The Role of Imaging Established and Emerging Non-mammalian Models in Neuroscience

Haynes

Ulland

Eliceiri

2022

Front. Mol. Neurosci.

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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.

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“…Early efforts to transduce this germ line had success rates akin to finding a needle in a haystack (7). Recent developments that isolate primordial germ cells from blood and then culture them have enabled lentiviral-driven genetic manipulation and selection with transformative success (8,9). Given the new insights into zebra finch LDLR, the efficiency of this process might be improved by differentially pseudo-typing the virus, to exploit more conserved entry portals.…”

mentioning

confidence: 99%

“…Given what we now know about the zebra finch LDLR, how did neural transduction and the resultant behavioral effects reported in these studies happen? While the canonical mammalian LDLR is missing, other members of the low-density lipoprotein receptor family are expressed in zebra finch tissue and could provide an alternative entry portal to cells (9). In addition to hijacking receptors, many viruses have generalized entry mechanisms.…”

mentioning

confidence: 99%

Why zebra finches don’t get hypercholesterolemia

White

Wright

2021

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

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In his 1994 classic, "Why Zebras Don't Get Ulcers"(1), Robert Sapolsky chronicles how the pathological effects of chronic stress commonly seen in humans are much rarer in non-human animals. A new paper by Velho et al. (2) in this issue of Proceedings of the National Academy of Sciences reports a fascinating discovery that provides an update to this theme. In this paper, the authors investigate the evolution of a receptor involved in cholesterol uptake by cells that is also exploited by the vesicular stomatitis virus (VSV) to enter cells during infection. They find that in songbirds-including the popular lab model, the zebra finch-this low-density lipid receptor (LDLR) is missing several key protein coding regions that are found in many other avian groups as well as in mammals. This apparent case of evolutionary loss of function raises interesting questions about how songbirds manage to balance their cholesterol levels and avoid hypercholesterolemia (hence the title of this commentary). It also has important implications for a variety of other fields, ranging from efforts to use VSV-G pseudotyped lentiviruses as a tool to manipulate gene expression, to efforts to understand and control vesicular stomatitis, a major agricultural disease caused by VSV.

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Highly Efficient Genome Modification of Cultured Primordial Germ Cells with Lentiviral Vectors to Generate Transgenic Songbirds

Cited by 15 publications

References 47 publications

Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata)

Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata)

A Model of Discovery: The Role of Imaging Established and Emerging Non-mammalian Models in Neuroscience

Why zebra finches don’t get hypercholesterolemia

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