Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2E450fs mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.
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This work is supported by the Alex's Lemonade Stand Foundation (J.S.B., M.S.I., D.M.L.), the Leukemia & Lymphoma Society (J.S.B.), the American Cancer Society (D.M.L.), a Massachusetts General Hospital Howard Goodman Fellowship (D.M.L.) and US National Institutes of Health grants F32DK098875 (F.E.M.), R24OD016761 and 1R01CA154923 (D.M.L.). Q.T. is funded by the China Scholarship Council. J.N.B. is funded as the Cancer Care Nova Scotia Peggy Davison Clinician. We thank D. Traver, T. North and J. Rawls for their helpful comments and B. Li for advice.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Homozygous rag2E450fs mutants are healthy and viable similar to wild-type and heterozygous siblings.
(left) Representative bright-field images of wild type (wt), heterozygous (het) and homozygous mutant (mut) animals derived from a rag2E450fs heterozygous mating. (right) Quantification of animal length at 2.5 months of age with number of genotyped animals per clutch annotated. Error bars show ±1 s.d. Scale bars denote 2.5 mm.
Supplementary Figure 2 Homozygous rag2E450fs mutants have reduced T-cell numbers and thymus size at 5 d of life.
(a-b) Whole mount in situ hybridization for rag1 in 5-day-old zebrafish, arrows denote thymus. (c) 2-dimensional pixel counts to quantify thymus size based on rag1 staining. Error bars denote ±1 standard deviation and asterisk denotes a significance of p=0.002, Student’s t-test.
Supplementary Figure 3 Percentage of ubi-EGFP+ blood cell engraftment in recipient fish at 45 d post transplantation.
The percentage of EGFP+ cells assessed by FACS of whole kidney marrow isolated from AB-strain wild type fish (Control) or transplanted wild type and homozygous rag2E450fs mutant zebrafish.
FACS analysis of whole blood isolated from AB-strain wild type fish (negative control) and stained with propidium iodide (PI), compared to manually dissected muscle obtained from 12-month-old mylpfa-EGFP transgenic animals.
Supplementary Figure 5 Homozygous rag2E450fs mutant zebrafish engraft EGFP+ muscle from ubi-EGFP transgenic donor animals.
(a) Low magnification images of rag2E450fs mutant zebrafish engrafted with ubi-EGFP donor muscle cells. Myotome segments (arrows), anti-GFP immunostaining (αGFP) and hematoxylin and eosin (H&E). (b) Higher magnification views of boxed region. Activated caspase 3 antibody (αCaspase3) and TUNEL staining on adjacent sections. (c) Thymus sections. Low magnification (left panel) and high magnification views of boxed region (right three panels). Hematoxylin and eosin stained sections (left two panels). Right two panels show a large number of thymic T cells undergo apoptosis during T cell maturation and providing a good control for verifying apoptotic cell staining on section. Scale bars denote 500 µm (a), 100 µm (b, c-left), 50 µm (c-three right panels).
(a) Genomic organization of the zebrafish rag2 gene with the core domain and Plant Homeodomain (PHD) denoted. Nucleotide sequences of wild type and mutant rag2E450fs alleles that span the ZFN target site (yellow), nucleotide additions blue and deletions gray. Boxed nucleotides denote de novo creation of an XcmI site within the rag2E450fs allele. Following PCR from fin clip isolated genomic DNA, amplified fragments are digested with XcmI and resolved on a 2% agarose gel containing ethidium bromide (b). Representative gel image with wild type (wt), heterozygous (het), and homozygous mutant (mut) animal results noted.
Supplementary Figures 1–6 and Supplementary Tables 1–6 (PDF 2410 kb)
Homozygous rag2E450fs mutant zebrafish engrafted with marrow from ubi-EGFP transgenic donors showing circulation of GFP+ blood cells in the tail at 30 d post-transplantation. (MOV 17271 kb)
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Tang, Q., Abdelfattah, N., Blackburn, J. et al. Optimized cell transplantation using adult rag2 mutant zebrafish. Nat Methods 11, 821–824 (2014). https://doi.org/10.1038/nmeth.3031
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