Allogeneic organ transplantation is a powerful tool for clinical and basic research studies. However, the graft is often rejected by the host organism. Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish. These zebrafish escaped rejection, which made it possible to successfully transplant fragments of an allogeneic testis and testicular hyperplasia. This protocol can be used to amplify and maintain testicular hyperplasia grafts for several years (Kawasaki et al., 2016). The amplified hyperplasias are likely to be a good source of somatic and germ cells such as Sertoli cells and spermatogonial stem cells.
[Background] Zebrafish have emerged as a tractable teleost genetic model for the study of vertebrate biology because several thousand mutants have been isolated by various genetic methods (Granato and Nüsslein-Volhard, 1996). Recently, this organism was used to study human diseases such as cancer (White et al., 2013). Although the incidence of spontaneous cancers is low, with many zebrafish eventually surviving cancer, allogeneic organ transplantation is a powerful tool, because many of the cancers are not syngeneic. Unfortunately, this method is not well developed. A previous study reported that zebrafish embryos accept cell grafts prior to the development of a mature immune system (Nicoli et al., 2007). However, it is difficult to successfully transplant grafts into embryos due to their minute size. For transplantation into adult zebrafish, sublethal γ-irradiation or immunosuppression with dexamethasone can block the rejection of the graft (Stoletov et al., 2007; White et al., 2008). However, it can be difficult to maintain cell grafts for long periods of time due to the short lifespans of recipients and the recovery of the immune response by 20 days after irradiation (Smith et al., 2010; Eguiara et al., 2011). Tissue grafts between identical clonal or inbred lines can survive without rejection (Kawasaki et al., 2010; Mizgirev and Revskoy, 2010; Shinya and Sakai, 2011).
T lymphocytes are central to the allograft response (Ingulli, 2010). The Recombination activating gene 1, 2 (rag1, Rag2) are important for immune function, because it creates double-stranded DNA breaks and is essential for V(D)J recombination, as well as for T and B cell function. rag1 mutant mice lack mature T and B cells, and they maintain allogeneic heart grafts for long periods of time (Zhang et al., 2006). By contrast, allogeneic transplantation has failed in rag1 mutant rats, probably due to the insufficient depletion of T and B cells (Ménoret et al., 2013). Hypomorphic rag2E450fs mutant zebrafish has been created, which have reduced V(D)J rearrangement and lymphocytes, and maintains various allogeneic cancer cells (Tang et al., 2014). Although rag1t26683 mutant zebrafish (hereafter rag1 mutant) have been isolated and they lack functional T and B cells (Wienholds et al., 2002; Petrie-Hanson et al., 2009), they were not used for transplantation. Our recent study reported that rag1 mutant zebrafish accept and maintain allogeneic testis organ and testicular hyperplasia grafts for long periods of time (Kawasaki et al., 2016). Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish for the subcutaneous transplantation of testis and testicular hyperplasia grafts.
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