Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Sconocchia, T; Foßelteder, J; Köhnke, T; Majeti, R; Reinisch, A.
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells.
J Vis Exp. 2023; (193): e64558
Doi: 10.3791/64558
Web of Science
PubMed
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- Führende Autor*innen der Med Uni Graz
- Reinisch Andreas
- Sconocchia Tommaso
- Co-Autor*innen der Med Uni Graz
- Foßelteder Johannes
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- Abstract:
- Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.
- Find related publications in this database (using NLM MeSH Indexing)
- Humans - administration & dosage
- Gain of Function Mutation - administration & dosage
- Gene Editing - methods
- Hematopoietic Stem Cells - administration & dosage
- Mutation - administration & dosage
- CRISPR-Cas Systems - administration & dosage