Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research
Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former...
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| Format: | Article |
| Language: | English |
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Wiley
2023-07-01
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| Series: | FEBS Open Bio |
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| Online Access: | https://doi.org/10.1002/2211-5463.13581 |
| _version_ | 1797789183563005952 |
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| author | Stefano L. Giandomenico Erin M. Schuman |
| author_facet | Stefano L. Giandomenico Erin M. Schuman |
| author_sort | Stefano L. Giandomenico |
| collection | DOAJ |
| description | Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise, and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e., knock‐down, knock‐out, mutation, and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination, and epigenetic silencing. The advent of CRISPR‐Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often, the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA‐based manipulation. Our aim is to present a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to providing the inexperienced reader with a conceptual framework on how to use these tools, and for more in‐depth information, we will provide specific references throughout. |
| first_indexed | 2024-03-13T01:47:06Z |
| format | Article |
| id | doaj.art-edee7048f439482d907aa88e5e58f645 |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-03-13T01:47:06Z |
| publishDate | 2023-07-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-edee7048f439482d907aa88e5e58f6452023-07-03T07:24:35ZengWileyFEBS Open Bio2211-54632023-07-011371164117610.1002/2211-5463.13581Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery researchStefano L. Giandomenico0Erin M. Schuman1Max Planck Institute for Brain Research Frankfurt am Main GermanyMax Planck Institute for Brain Research Frankfurt am Main GermanyGaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise, and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e., knock‐down, knock‐out, mutation, and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination, and epigenetic silencing. The advent of CRISPR‐Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often, the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA‐based manipulation. Our aim is to present a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to providing the inexperienced reader with a conceptual framework on how to use these tools, and for more in‐depth information, we will provide specific references throughout.https://doi.org/10.1002/2211-5463.13581CRISPRdeliverydifferentiationneuroscienceTPD |
| spellingShingle | Stefano L. Giandomenico Erin M. Schuman Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research FEBS Open Bio CRISPR delivery differentiation neuroscience TPD |
| title | Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research |
| title_full | Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research |
| title_fullStr | Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research |
| title_full_unstemmed | Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research |
| title_short | Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research |
| title_sort | genetic manipulation and targeted protein degradation in mammalian systems practical considerations tips and tricks for discovery research |
| topic | CRISPR delivery differentiation neuroscience TPD |
| url | https://doi.org/10.1002/2211-5463.13581 |
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