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换一换Generating CRISPR-Cas9-Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells
Oliver J. Bower, Afshan McCarthy, Rebecca A. Lea, Gregorio Alanis-Lobato, Jasmin Zohren, Claudia Gerri, James M.A. Turner, Kathy K. Niakan, Oliver J. Bower, Afshan McCarthy, Rebecca A. Lea, Gregorio Alanis-Lobato, Jasmin Zohren, Claudia Gerri, James M.A. Turner, Kathy K. Niakan
CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. **Basic Protocol 1** : Selecting and ligating sgRNAs into expression plasmids **Basic Protocol 2** : Validation of sgRNA via _in vitro_ transcription and cleavage assay **Basic Protocol 3** : Nucleofection of primed human embryonic stem cells **Basic Protocol 4** : MiSeq analysis of indel mutations **Basic Protocol 5** : Single cell cloning of targeted hPSCs **Basic Protocol 6** : Karyotyping of targeted hPSCs
AI 解读Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses
David Vandael, Yuji Okamoto, Carolina Borges-Merjane, Victor Vargas-Barroso, Benjamin A. Suter, Peter Jonas
Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre–postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system.
AI 解读Stem-cell-derived human microglia transplanted into mouse brain to study human disease
Nicola Fattorelli, Anna Martinez-Muriana, Leen Wolfs, Ivana Geric, Bart De Strooper, Renzo Mancuso
Microglia are critically involved in complex neurological disorders with a strong genetic component, such as Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia. Although mouse microglia can recapitulate aspects of human microglia physiology, they do not fully capture the human genetic aspects of disease and do not reproduce all human cell states. Primary cultures of human microglia or microglia derived from human induced pluripotent stem cells (PSCs) are difficult to maintain in brain-relevant cell states in vitro. Here we describe MIGRATE (microglia in vitro generation refined for advanced transplantation experiments, which provides a combined in vitro differentiation and in vivo xenotransplantation protocol to study human microglia in the context of the mouse brain. This article details an accurate, step-by-step workflow that includes in vitro microglia differentiation from human PSCs, transplantation into the mouse brain and quantitative analysis of engraftment. Compared to current differentiation and xenotransplantation protocols, we present an optimized, faster and more efficient approach that yields up to 80% chimerism. To quantitatively assess engraftment efficiency by flow cytometry, access to specialized flow cytometry is required. Alternatively, the percentage of chimerism can be estimated by standard immunohistochemical analysis. The MIGRATE protocol takes ~40 d to complete, from culturing PSCs to engraftment efficiency assessment.
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