Simultaneous visualization of multiple neuronal properties with single-cell resolution in the living rodent brain

Ako, Rie; Wakimoto, Mayu; Ebisu, Haruka; Tanno, Kaori; Hira, Riichiro; Kasai, Hideaki; Matsuzaki, Masunori; Kawasaki, Hiroshi

doi:10.1016/j.mcn.2011.08.005

Cited by 39 publications

(52 citation statements)

References 49 publications

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“…1C, D). These observations indicate that selective unilateral labeling of layer 2/3 neurons can be efficiently achieved using in utero electroporation at E18.0, and are consistent with previous reports using mice (Saito and Nakatsuji, 2001;Tabata and Nakajima, 2001;Sehara et al, 2010;Ako et al, 2011).…”

Section: Selective Unilateral Labeling Of Layer 2/3 Neurons In the Rasupporting

confidence: 92%

“…pCAG-EGFP was as described previously (Sehara et al, 2010). pCAG-synaptophysin-mCherry was constructed by substituting EGFP of pCAG-synaptophysin-EGFP (Sehara et al, 2010;Ako et al, 2011) with mCherry. Plasmids were purified using the Endofree plasmid maxi kit (Qiagen, Valencia, OR).…”

Section: Plasmidsmentioning

confidence: 99%

“…In utero electroporation was performed as described previously with slight modifications (Sehara et al, 2010;Ako et al, 2011). Briefly, pregnant rats (E18.0) and mice (E15.5) were anesthetized with sodium pentobarbital, and the uterine horns were exposed.…”

Section: In Utero Electroporationmentioning

confidence: 99%

“…Furthermore, using in utero electroporation, neurons can be transfected with multiple genes, such as the presynaptic marker synaptophysin-mCherry and the postsynaptic marker PSD-95-GFP. Simultaneous investigation of axonal trajectories and functional properties of a selected subset of neurons in vivo by combining in utero electroporation and these marker proteins (Ako et al, 2011) would contribute toward an understanding of the structure and function of intracortical circuitry.…”

Section: In Utero Electroporation For the Analysis Of Neuronal Circuitrymentioning

confidence: 99%

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Distinct developmental principles underlie the formation of ipsilateral and contralateral whisker-related axonal patterns of layer 2/3 neurons in the barrel cortex

et al. 2012

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Section: Selective Unilateral Labeling Of Layer 2/3 Neurons In the Rasupporting

confidence: 92%

Section: Plasmidsmentioning

confidence: 99%

Section: In Utero Electroporationmentioning

confidence: 99%

Section: In Utero Electroporation For the Analysis Of Neuronal Circuitrymentioning

confidence: 99%

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Distinct developmental principles underlie the formation of ipsilateral and contralateral whisker-related axonal patterns of layer 2/3 neurons in the barrel cortex

et al. 2012

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“…pCAG-EGxxFP-Satb2 contains a genomic fragment including the sgRNA target sequence deliver transgenes into the living rodent brain [7][8][9][10][11][12] , here we report a brain-specific gene knockout method, and demonstrate the power of our simple electroporation-based gene knockout in the living mouse brain.…”

mentioning

confidence: 75%

CRISPR/Cas9‐Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation

Shinmyo

Kawasaki

2017

CP Neuroscience

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The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo.Technologies for disrupting the genome in living animals are essential for elucidating the physiological mechanisms of our body and the pathological mechanisms of diseases, and for contributing to the discovery of new therapeutic interventions for human diseases. One technique which has been widely utilized is homologous recombination-based gene targeting in embryonic stem (ES) cells 1 . In addition, conditional mutagenesis using ES cells that relies on the DNA recombinase Cre and its recognition site loxP has become a valuable tool to achieve gene targeting in selected cell types 2 . Although this gene targeting technology using ES cells is extensively utilized for generating various kinds of genetically modified mice, it is relatively arduous, costly and time-consuming.Recently, a simple and efficient genome targeting technology has been developed based on the microbial type II clustered, regularly interspaced, short palindromic repeats (CRISPR)/associated protein (Cas) adaptive immune systems derived from Streptococcus pyogenes 3 . Cas9 endonuclease guided by a duplex of mature CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) cleaves trespassing DNA from bacteriophages or plasmids in a sequence-specific manner 3 . In mammalian cells, it has been demonstrated that a combination of Cas9 and sgRNA, which is an artificial chimera of crRNA and tracrRNA, caused site-specific DSBs, and as a result, genome targeting via insertions and deletions (indels) was caused by error-prone non-homologous end-joining (NHEJ) 4,5 . These features of the CRISPR/Cas9 system facilitated the generation of mice carrying mutations in specific genes 6 . Indeed,...

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Inhibitory neuron‐specific Cre‐dependent red fluorescent labeling using VGAT BAC‐based transgenic mouse lines with identified transgene integration sites

Kaneko

Takatsuru

Morita

et al. 2017

J of Comparative Neurology

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Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.

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Simultaneous visualization of multiple neuronal properties with single-cell resolution in the living rodent brain

Cited by 39 publications

References 49 publications

Distinct developmental principles underlie the formation of ipsilateral and contralateral whisker-related axonal patterns of layer 2/3 neurons in the barrel cortex

Distinct developmental principles underlie the formation of ipsilateral and contralateral whisker-related axonal patterns of layer 2/3 neurons in the barrel cortex

CRISPR/Cas9‐Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation

Inhibitory neuron‐specific Cre‐dependent red fluorescent labeling using VGAT BAC‐based transgenic mouse lines with identified transgene integration sites

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