| Autor: |
Kaltenbacher, Thorsten, Löprich, Jessica, Maresch, Roman, Weber, Julia, Müller, Sebastian, Oellinger, Rupert, Groß, Nina, Griger, Joscha, de Andrade Krätzig, Niklas, Avramopoulos, Petros, Ramanujam, Deepak, Brummer, Sabine, Widholz, Sebastian A., Bärthel, Stefanie, Falcomatà, Chiara, Pfaus, Anja, Alnatsha, Ahmed, Mayerle, Julia, Schmidt-Supprian, Marc, Reichert, Maximilian, Schneider, Günter, Ehmer, Ursula, Braun, Christian J., Saur, Dieter, Engelhardt, Stefan, Rad, Roland |
| Zdroj: |
Nature Protocols; April 2022, Vol. 17 Issue: 4 p1142-1188, 47p |
| Abstrakt: |
Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3–5 weeks. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
Full text from SpringerLink