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J Exp Med. 2016 Nov 14;213(12):2575-2589. Epub 2016 Oct 24.

Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.

Moore JC1,2,3,4, Tang Q1,2,3,4, Yordán NT4,5, Moore FE1,2,3,4, Garcia EG1,2,3,4, Lobbardi R1,2,3,4, Ramakrishnan A1,2,3,4, Marvin DL2, Anselmo A6,7, Sadreyev RI6,7, Langenau DM8,2,3,4.

Author information

1
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.
2
Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.
3
Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.
4
Harvard Stem Cell Institute, Cambridge, MA 02139.
5
Harvard University, Cambridge, MA 02138.
6
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.
7
Department of Genetics, Harvard Medical School, Boston, MA 02115.
8
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129 dlangenau@mgh.harvard.edu.

Abstract

Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.

PMID:
27810924
PMCID:
PMC5110017
DOI:
10.1084/jem.20160378
[Indexed for MEDLINE]
Free PMC Article

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