Production of knockout mouse lines with Cas9
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved..
Knockout mice are used extensively to explore the phenotypic effects of mammalian gene dysfunction. With the application of RNA-guided Cas9 nuclease technology for the production of knockout mouse lines, the time, as well as the resources needed, to progress from identification of a gene of interest to production of a knockout line is significantly reduced. Here we present our standard methodology to produce knockout mouse lines by the electroporation of Cas9 ribonucleoprotein (RNP) into mouse zygotes. Using this protocol, we have obtained an 80% success rate in the generation of founders for null alleles with a subsequent 93% germline transmission rate. These methods rely on equipment already present in the majority of transgenic facilities and should be straightforward to implement where appropriate embryo handling expertise exists.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:191 |
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| Enthalten in: |
Methods (San Diego, Calif.) - 191(2021) vom: 05. Juli, Seite 32-43 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Gertsenstein, Marina [VerfasserIn] |
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| Links: |
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| Themen: |
EC 3.1.- |
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| Anmerkungen: |
Date Completed 04.01.2022 Date Revised 21.02.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ymeth.2021.01.005 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Knockout mice are used extensively to explore the phenotypic effects of mammalian gene dysfunction. With the application of RNA-guided Cas9 nuclease technology for the production of knockout mouse lines, the time, as well as the resources needed, to progress from identification of a gene of interest to production of a knockout line is significantly reduced. Here we present our standard methodology to produce knockout mouse lines by the electroporation of Cas9 ribonucleoprotein (RNP) into mouse zygotes. Using this protocol, we have obtained an 80% success rate in the generation of founders for null alleles with a subsequent 93% germline transmission rate. These methods rely on equipment already present in the majority of transgenic facilities and should be straightforward to implement where appropriate embryo handling expertise exists | ||
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