CRISPR‐Cas9‐based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR‐Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2′‐
Isolation and maintenance of in vitro cell cultures from the ctenophore Mnemiopsis leidyi
The ability to isolate, monitor, and examine specific cells of interest enables targeted experimental manipulations that would otherwise be difficult to perform and interpret in the context of the whole organism. In vitro primary cell cultures derived from ctenophores thus serve as an important tool for understanding complex cellular and molecular interactions that take place both within and between various ctenophore cell types. Here we describe methods for reliably generating and maintaining primary cell cultures derived from the lobate ctenophore Mnemiopsis leidyi that can be used for a wide variety of experimental applications.
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- Award ID(s):
- 2013692
- NSF-PAR ID:
- 10348360
- Editor(s):
- Blanchoud S.; Galliot B.
- Date Published:
- Journal Name:
- Methods in molecular biology
- Volume:
- 2450
- ISSN:
- 1064-3745
- Page Range / eLocation ID:
- 347-358
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract O ‐methyl‐3′phosphorothioate‐modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion‐deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient‐derived glioblastoma (GBM) stem‐like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem‐like cells. © 2020 Wiley Periodicals LLCBasic Protocol : Cas9:sgRNA ribonucleoprotein nucleofection for insertion‐deletion (indel) mutation and genomic deletion generation in primary cell cultures -
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Cell suspension fluidics, such as flow cytometry (FCS) and fluorescence-activated cell sorting (FACS), facilitates the identification and precise separation of individual cells based on phenotype. Since its introduction, flow cytometry has been used to analyze cell types and cellular processes in diverse non-vertebrate taxa, including cnidarians, molluscs, and arthropods. Ctenophores, which diverged very early from the metazoan stem lineage, have emerged as an informative clade for the study of metazoan cell type evolution. We present standardized methodologies for flow cytometry-mediated identification and analyses of cells from the model ctenophore
Mnemiopsis leidyi that can also be applied to isolate targeted cell populations. Here we focus on the identification and isolation of ctenophore phagocytes. Implementing flow cytometry methods in ctenophores allows for fine scale analyses of fundamental cellular processes conserved broadly across animals, as well as potentially revealing novel cellular phenotypes and behaviors restricted to the ctenophore lineage. -
null (Ed.)In bilaterians and cnidarians, epithelial cell-polarity is regulated by the interactions between Par proteins, Wnt/PCP signaling pathway, and cell-cell adhesion. Par proteins are highly conserved across Metazoa, including ctenophores. But strikingly, ctenophore genomes lack components of the Wnt/PCP pathway and cell-cell adhesion complexes raising the question if ctenophore cells are polarized by mechanisms involving Par proteins. Here, by using immunohistochemistry and live-cell imaging of specific mRNAs, we describe for the first time the subcellular localization of selected Par proteins in blastomeres and epithelial cells during the embryogenesis of the ctenophore Mnemiopsis leidyi . We show that these proteins distribute differently compared to what has been described for other animals, even though they segregate in a host-specific fashion when expressed in cnidarian embryos. This differential localization might be related to the emergence of different junctional complexes during metazoan evolution.more » « less
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Abstract Cnidarians are emerging model organisms for cell and molecular biology research. However, successful cell culture development has been challenging due to incomplete tissue dissociation and contamination. In this report, we developed and tested several different methodologies to culture primary cells from all tissues of two species of Cnidaria: Nematostella vectensis and Pocillopora damicornis . In over 170 replicated cell cultures, we demonstrate that physical dissociation was the most successful method for viable and diverse N. vectensis cells while antibiotic-assisted dissociation was most successful for viable and diverse P. damicornis cells. We also demonstrate that a rigorous antibiotic pretreatment results in less initial contamination in cell cultures. Primary cultures of both species averaged 12–13 days of viability, showed proliferation, and maintained high cell diversity including cnidocytes, nematosomes, putative gastrodermal, and epidermal cells. Overall, this work will contribute a needed tool for furthering functional cell biology experiments in Cnidaria.more » « less
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Despite long-standing experimental interest in ctenophores due to their unique biology, ecological influence and evolutionary status, previous work has largely been constrained by the periodic seasonal availability of wild-caught animals and difficulty in reliably closing the life cycle. To address this problem, we have developed straightforward protocols that can be easily implemented to establish long-term multigenerational cultures for biological experimentation in the laboratory. In this protocol, we describe the continuous culture of the Atlantic lobate ctenophore Mnemiopsis leidyi. A rapid 3-week egg-to-egg generation time makes Mnemiopsis suitable for a wide range of experimental genetic, cellular, embryological, physiological, developmental, ecological and evolutionary studies. We provide recommendations for general husbandry to close the life cycle of Mnemiopsis in the laboratory, including feeding requirements, light-induced spawning, collection of embryos and rearing of juveniles to adults. These protocols have been successfully applied to maintain long-term multigenerational cultures of several species of pelagic ctenophores, and can be utilized by laboratories lacking easy access to the ocean. We also provide protocols for targeted genome editing via microinjection with CRISPR–Cas9 that can be completed within ~2 weeks, including single-guide RNA synthesis, early embryo microinjection, phenotype assessment and sequence validation of genome edits. These protocols provide a foundation for using Mnemiopsis as a model organism for functional genomic analyses in ctenophores.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/978-1-0716-2172-1_18
