Highly efficient manipulation of nervous system gene expression with NEPTUNE

Mangold, Katrin; Mašek, J.; He, Jiehua; Lendahl, Urban; Fuchs, Elaine; Andersson, Emma R.

doi:10.1016/j.crmeth.2021.100043

Cited by 4 publications

(9 citation statements)

References 48 publications

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“…Currently, available approaches include the microinjection of viruses; however, this yields variable outcomes that are influenced by tropism and off-target insertions ( Artegiani and Calegari, 2013 ; Mangold et al, 2021 ). Alternatively, targeted electroporation of DNA constructs can produce consistent and spatially restricted genetic changes ( Pierreux et al, 2005 ; Mazari et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

Cre recombinase microinjection for single-cell tracing and localised gene targeting

et al. 2023

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Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.

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Section: Resultsmentioning

confidence: 99%

Cre recombinase microinjection for single-cell tracing and localised gene targeting

et al. 2023

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“…Genomic manipulation allows the precise expression of a gene of interest in a temporally and spatially regulated manner; however, it requires a specific Cre-expressing mouse strain or knowledge of specific distal elements to control expression, and a significant investment of time, cost, and space to establish and maintain a transgenic mouse strain. The NEPTUNE method, which uses the ultrasound-guided injection of viruses, addresses many weaknesses of genomic manipulation and allows for the analysis of a gene of interest in less time, space, and animals 17 . However, it still requires specific equipment, such as ultrasound microscopy.…”

Section: Discussionmentioning

confidence: 99%

“…For analyzing the role of genes of interest at E7 or E8, a period of rapid organ development, ultrasound-guided method is useful to introduce viral vectors into embryos. Although previous methods for E8.5 embryos resulted in low survival rates and abnormalities in many surviving embryos 16 , a recent method for E7.5 embryos, called NEPTUNE, greatly improved the survival rate of embryos 17 . As the neural tube is still open at E7.5, a viral vector injected into the amniotic fluid can access the neuroepithelial cells in addition to the epithelial and endodermal tissues 17 .…”

Section: Introductionmentioning

confidence: 99%

“…Although previous methods for E8.5 embryos resulted in low survival rates and abnormalities in many surviving embryos 16 , a recent method for E7.5 embryos, called NEPTUNE, greatly improved the survival rate of embryos 17 . As the neural tube is still open at E7.5, a viral vector injected into the amniotic fluid can access the neuroepithelial cells in addition to the epithelial and endodermal tissues 17 . The high efficiency of gene transduction achieved by the NEPTUNE-mediated knockdown of Olig2 or Sptbn2 resulted in phenotypes similar to those of their knockout embryo counterparts 17 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure

Kuwayama

Kishi

Maeda

et al. 2020

Preprint

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22During the early stage of mammalian neural development, neuroepithelial cells (NECs) 23 proliferate and increase their pool size before switching their fate to become neurogenic 24 radial glial progenitors (RGPs). The timing of this expansion-to-neurogenic phase 25 transition is strictly regulated so as to determine the proper number of progenitors and 26 differentiated cell types that constitute the brain. The molecular mechanism underlying 27 this switch has remained poorly understood, however, in part because of the difficulty 28 associated with manipulation of gene expression in NECs before their transition to 29 RGPs, which occurs before neural tube closure. We have now developed a simple and 30 efficient method to manipulate gene expression in mouse neocortical NECs in the 31 expansion phase by in utero injection of viral vectors at embryonic day 7.0 to 8.0. With 32 the use of this method, we found that knockdown of the chromatin-associated protein 33Hmga2 in NECs inhibited the onset of the neurogenic phase in the neocortex. This 34 effect of Hmga2 knockdown was accompanied by inhibition of the expression of a set 35 of genes targeted by Polycomb group proteins, which repress neurogenic genes in 36 cortical progenitors. Our study thus establishes a novel method for manipulation of gene 37 expression in the early stage of mouse brain development as well as uncovers a key 38 molecular player in the generation of neurogenic progenitors in the developing mouse 39 neocortex. 40 41 E8.0. This method is based on in utero injection of viral vectors into amniotic fluid and 130 is associated with a high survival rate without inducing gross developmental 131 abnormalities. Moreover, as a proof of concept, we have identified a key regulator of 132 the E-to-N transition with the use of this new method. We found that Hmga2 expression 133 in neocortical NPCs peaks at around the E-to-N transition (at E10), and that knockdown 134 of Hmga2 from E8.0 delayed the onset of neurogenesis and shifted the transcriptional 135 profile of NPCs at this transition closer to that in the expansion phase. Importantly, 136 knockdown of Hmga2 from E8.0 attenuated the expression of a subset of PcG target 137 genes in NPCs at E11.0. Our results suggest that an increase in Hmga2 expression 138 triggers the onset of the neurogenic phase in part through activation of neurogenesis-139

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“…In a similar vein, researchers targeting the mice subplate neurons born around E10 face a similar difficulty localizing the embryo ( Ohtaka-Maruyama et al, 2018 ). The ultrasound guided electroporation approach used in Neural Plate Targeting within Utero Nano-Injection (NEPTUNE) for lentiviral injections at E7.5, could be modified for IUE to aid in localizing the ventricles ( Mangold et al, 2021 ). Given the popularity of IUE as a technique, it is no wonder that improvements to the technical aspects of IUE, as well as the tools driving exogenous expression of the GOI are being developed at such breakneck speeds.…”

Section: Perspectives and Concluding Remarksmentioning

confidence: 99%

Visualizing Cortical Development and Evolution: A Toolkit Update

Kumamoto

Ohtaka-Maruyama

2022

Front. Neurosci.

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Visualizing the process of neural circuit formation during neurogenesis, using genetically modified animals or somatic transgenesis of exogenous plasmids, has become a key to decipher cortical development and evolution. In contrast to the establishment of transgenic animals, the designing and preparation of genes of interest into plasmids are simple and easy, dispensing with time-consuming germline modifications. These advantages have led to neuron labeling based on somatic transgenesis. In particular, mammalian expression plasmid, CRISPR-Cas9, and DNA transposon systems, have become widely used for neuronal visualization and functional analysis related to lineage labeling during cortical development. In this review, we discuss the advantages and limitations of these recently developed techniques.

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Highly efficient manipulation of nervous system gene expression with NEPTUNE

Cited by 4 publications

References 48 publications

Cre recombinase microinjection for single-cell tracing and localised gene targeting

Cre recombinase microinjection for single-cell tracing and localised gene targeting

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure

Visualizing Cortical Development and Evolution: A Toolkit Update

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