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Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Authors
  • Sano, Soichi1
  • Wang, Ying1
  • Evans, Megan A.1
  • Yura, Yoshimitsu1
  • Sano, Miho1
  • Ogawa, Hayato1
  • Horitani, Keita1
  • Doviak, Heather1
  • Walsh, Kenneth1
  • 1 Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine
Type
Published Article
Journal
Journal of Visualized Experiments
Publisher
MyJoVE Corporation
Publication Date
Oct 03, 2019
Issue
152
Identifiers
DOI: 10.3791/59977
PMID: 31633690
PMCID: PMC7249700
Source
PubMed Central
Keywords
License
Unknown

Abstract

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

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