Supplementary Figure 1. Homozygous rag2 E450fs mutants are healthy and viable similar to wild-type and heterozygous siblings.

Transcription

1 Supplementary Figure 1 Homozygous rag2 E450fs 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 rag2 E450fs 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.

2 Supplementary Figure 2 Homozygous rag2 E450fs 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.

3 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 rag2 E450fs mutant zebrafish.

4 Supplementary Figure 4 Manual dissection and cell harvesting protocols produce viable muscle cells. 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.

5 Supplementary Figure 5 E450fs Homozygous rag2 animals. mutant zebrafish engraft EGFP+ muscle from ubi-egfp transgenic donor (a) Low magnification images of rag2e450fs mutant zebrafish engrafted with ubi-egfp donor muscle cells. Myotome segments (arrows), anti-gfp immunostaining ( GFP) and hematoxylin and eosin (H&E). (b) Higher magnification views of boxed region. Activated caspase 3 antibody ( Caspase3) 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).

6 Supplementary Figure 6 Genotyping strategy to identify rag2 E450fs mutants. (a) Genomic organization of the zebrafish rag2 gene with the core domain and Plant Homeodomain (PHD) denoted. Nucleotide sequences of wild type and mutant rag2 E450fs 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 rag2 E450fs 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.

7 Supplementary Table 1 Lay rates for mass matings of heterozygous (+/-) and homozygous mutant (-/-) rag2 E450fs zebrafish (3 males and 3 females per breeding clutch).

8 Supplementary Table 2 Analysis of blood cell types in rag2 E450fs mutant (-/-) and wild-type sibling (+/+) animals.

9 Supplementary Table 3 Engraftment rates for animals transplanted with hematopoietic stem cells and muscle stem cells.

10 Supplementary Table 4 Engraftment rates for animals transplanted with transgenic zebrafish tumors.

11 Supplementary Table 5 Quantitative real time PCR primers to assess gene expression.

12 Supplementary Table 6 PCR primers to assess and igm receptor rearrangements.