Generation of mouse models carrying B cell restricted single or multiplexed loss-of-function mutations through CRISPR-Cas9 gene editing

Elisa Ten Hacken; Michaela Gruber; María Hernández-Sánchez; Gabriela Brunsting Hoffmann; Kaitlyn Baranowski; Robert A Redd; Kendell Clement; Kenneth Livak; Catherine J Wu

doi:10.1016/j.xpro.2023.102165

Generation of mouse models carrying B cell restricted single or multiplexed loss-of-function mutations through CRISPR-Cas9 gene editing

STAR Protoc. 2023 Dec 15;4(4):102165. doi: 10.1016/j.xpro.2023.102165. Epub 2023 Sep 18.

Authors

Elisa Ten Hacken¹, Michaela Gruber², María Hernández-Sánchez³, Gabriela Brunsting Hoffmann⁴, Kaitlyn Baranowski⁴, Robert A Redd⁵, Kendell Clement⁶, Kenneth Livak⁷, Catherine J Wu⁸

Affiliations

¹ Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA. Electronic address: elt4010@med.cornell.edu.
² CEMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
³ Department of Biochemistry and Molecular Biology, Pharmacy School, Universidad Complutense de Madrid, Madrid, Spain.
⁴ Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA.
⁵ Department of Data Science, Dana Farber Cancer Institute, Boston, MA, USA.
⁶ Broad Institute of MIT and Harvard, Cambridge, MA, USA; Molecular Pathology Unit, Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
⁷ Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA; Translational Immunogenomics Laboratory, Dana Farber Cancer Institute, Boston, MA, USA.
⁸ Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Electronic address: cwu@partners.org.

Abstract

Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitro transduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders. For complete details on the use and execution of this protocol, please refer to ten Hacken et al.,¹ ten Hacken et al.,² and ten Hacken et al.³.

Keywords: CRISPR; Cancer; Cell Biology; Cell Isolation; Genetics; Health Sciences; Immunology; Model Organisms; Molecular Biology.

MeSH terms

Animals
CRISPR-Cas Systems / genetics
Disease Models, Animal
Gene Editing* / methods
Lymphoproliferative Disorders* / genetics
Mice
Mutation
RNA, Guide, CRISPR-Cas Systems

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

MeSH terms

Substances

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