Optimized cell transplantation using adult rag2 mutant zebrafish

Abstract

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) 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. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

Figure 1. rag2^E450fs mutant zebrafish lack mature T cells and have a reduced B cell repertoire

(a) Human RAG2 protein with known SCID mutations denoted. Arrow denotes ZFN target region. (b) Nucleotide (top) and protein sequence (bottom) for the rag2^E450fs mutation. Yellow denotes ZFN target sites, blue nucleotide additions, gray nucleotide deletions, and arrow amino acid change at E450. Underlining shows amino acid sequence following frame shift with termination amino acid numbered. (c,d) Thymus sections of 90-day-old wild type (c) and rag2^E450fs mutant zebrafish (d). Red dashed lines denote thymus (left). Right panels are amplified views of boxed regions with adipocytes (A) and vacant thymic epithelium (E) shown. (e,f) Whole kidney marrow cytospins of wild type (e) and rag2^E450fs zebrafish (f) with lymphocytes (black arrows), erythrocytes (magenta arrows) and granulocytes (cyan arrows) denoted. Scale bars are 200 μm (c,d – left); 50 μm (c,d – right); 20 μm (e,f). (g) Cell counts from cytospins performed on whole kidney marrow. Error bars represent ± 1 s.d., p < 0.05 by Student’s t-test. (h) Gene expression analysis of whole kidney marrow cells. Error bars represent s.e.m, p < 0.05 by Student’s t-test. (i) PCR analysis for tcrb and igm rearrangement of whole kidney marrow cells from wild type (wt) and rag2^E450fs zebrafish. negative control (neg).

Figure 2. rag2^E450fs mutant fish engraft hematopoietic and muscle stem cells

(a) ubi-EGFP transgenic donor fish. (b–c) Wild type (wt, b) or homozygous rag2^E450fs recipient fish (c) transplanted with GFP-labeled marrow and imaged at 45 days post-transplantation. Fluorescent image of whole fish (left) with engraftment rates noted. FACS (middle) and cytospin analysis (right) of whole kidney marrow from donor (a) and recipient fish (b–c). FACS sorted GFP+ cells are shown for cytospin analysis in c. Lymphocytes (black arrows), red blood cells (magenta arrows), granulocytes (cyan arrows). (d–e) Fluorescent image of wild type (wt) and rag2^E450fs mutant fish engrafted with muscle cells from ubi-EGFP transgenic fish imaged at 30 days post-transplantation. (f) rag2^E450fs fish engrafted with muscle cells from alpha-actin-RFP transgenic fish at 30 days post-transplantation. Engraftment rates for d–f are shown on magnified image panels to the right. Scale bars are 2 mm.

Figure 3. Engraftment of zebrafish tumors into rag2^E450fs mutant fish

(a–d) Donor tumors used in cell transplantation studies. dsRED-labeled Myc-induced T-ALL arising in TuAB strain fish (a), zsYellow-labeled T-ALL from CG1-strain fish (b), GFP-labeled embryonal rhabdomyosarcoma (ERMS) from CG1-strain fish (c), and mitfa and BRAF^V600E induced melanoma arising in p53-deficient nacre strain fish (d). Merged bright-field and fluorescent images of wild type (e–h) or rag2^E450fs mutant fish (i–l) at 30 days post-transplantation. Images of whole kidney marrow sections (e,i), peripheral blood cytospins (f,j), and skeletal muscle (g,k,h,i). Red blood cells denoted by magenta arrows and renal tubules white arrows (e). Scale bars in fish images are 2 mm and histopathology images are 25 μm (e,i), 20 μm (f,j), and 50 μm (g,k,h,l).