Optimized cell transplantation using adult rag2 mutant zebrafish

Tang, Qin; Abdelfattah, Nouran S.; Blackburn, Jessica S.; Moore, John C.; Martinez, Sarah; Moore, Finola; Lobbardi, Riadh; Tenente, Inês M.; Ignatius, Myron S.; Berman, Jason N.; Liwski, Robert; Houvras, Yariv; Langenau, David M.

doi:10.1038/nmeth.3031

Cited by 130 publications

(131 citation statements)

References 24 publications

Supporting

Mentioning

125

Contrasting

Order By: Relevance

“…The rag1 (17) and mpx (18) Histology and thymus size quantification. Thymus histology was performed as previously described (21). Briefly, 3-month-old fish were sacrificed and fixed in 4% paraformaldehyde, embedded in paraffin, and step sectioned.…”

Section: Generation Of Zap70mentioning

confidence: 99%

“…Upon extraction, RNA was made into cDNA using SuperScript III reverse transcriptase (Invitrogen; 18080-051). tcrb and igm receptor rearrangements were PCR amplified using a nested protocol, as previously described (21). The first PCR utilized 2 ng of cDNA and was performed using an optimized 23.75-l reaction mixture with Choice Taq Blue master mix (Denville Scientific; CB4065-8).…”

Section: Generation Of Zap70mentioning

confidence: 99%

See 1 more Smart Citation

T Cell Immune Deficiency in zap70 Mutant Zebrafish

Moore

Mulligan

Yordán

et al. 2016

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

j ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP ؉ thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency. Z AP70 [zeta-chain (TCR) associated protein kinase, 70 kDa], is an important mediator of T cell differentiation and function. ZAP70 is a cytoplasmic protein kinase consisting of two aminoterminal Src homology 2 (SH2) domains and a carboxy-terminal kinase domain that plays a key role in regulating the T cell receptor (TCR) phosphorylation cascade. TCRs are activated by interacting with peptide antigens bound to major histocompatibility complexes (MHCs), causing the recruitment of CD4 or CD8 costimulatory receptors. The CD4 and CD8 receptors are bound by intracellular lymphocyte-specific protein kinase (LCK), which, in turn, phosphorylates the TCR immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3. Phosphorylated ITAMs recruit ZAP70 by binding its SH2 domains with high affinity. LCK phosphorylates ITAM-bound ZAP70, releasing it from an autoinhibited conformation and promoting its catalytic activity. Further phosphorylation of the TCR-associated scaffolding proteins by ZAP70 allows for the recruitment of additional signaling molecules and ultimately results in T cell activation, proliferation, and differentiation (1).Human ZAP70 deficiencies result in severe combined immune deficiency (SCID) with specific defects in T cell maturation (2-4). Patients with inactivating mutations in ZAP70 lack mature CD8 ϩ cytotoxic T cells and produce nonfunctional CD4 ϩ helper T cells. ZAP70 null CD4 ϩ T cells exit the thymus, yet they have dysfunctional T cell signaling and cannot mount effective T cell responses. Zap70 mutant mice also have T cell deficiencies, but they exhibit key differences compared with humans (5, 6). Zap70 mutant mice have a more severe block in thymocyte maturation, with T cells arresting at the CD4 ϩ /CD8 ϩ corti...

show abstract

Section: Generation Of Zap70mentioning

confidence: 99%

Section: Generation Of Zap70mentioning

confidence: 99%

T Cell Immune Deficiency in zap70 Mutant Zebrafish

Moore

Mulligan

Yordán

et al. 2016

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Because generation of mature B and T cells requires the activities of the RAG (recombination-activating gene) proteins, one way to alleviate rejection is to use mutations in the rag genes (Belizario, 2009;Tang et al, 2014;Tenente et al, 2014). As the host of transplantation we chose rag1 t26683 mutant zebrafish (hereafter rag1 mutant), which harbors a nonsense mutation in the catalytic domain of the encoded RAG1 protein resulting in loss of mature functional T and B cells (Wienholds et al, 2002;PetrieHanson et al, 2009).…”

Section: Transplantation Of a Testicular Hyperplasia Into Rag1 Mutantmentioning

confidence: 99%

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

2015

View full text Add to dashboard Cite

Molecular dissection and chemical screening on a complex process such as spermatogenesis could be facilitated by cell culture approaches that allow easy access for experimental manipulation and live imaging of specific molecules; however, technical limitations have thus far prevented the complete reconstruction of spermatogenic events in cell culture. Here, we describe the production of functional sperm from self-renewing spermatogonial stem cells (SSCs) in cell culture conditions, using zebrafish testicular hyperplasia cells that accumulate early stage spermatogonia. By serially transplanting hyperplasias into immunodeficient rag1 mutant zebrafish, we succeeded in long-term maintenance and efficient production of starting material for SSC culture. Through improvements of culture conditions, we achieved efficient propagation of SSCs derived from the hyperplasia. When SSCs that underwent the SSC-propagating step for 1 month were transferred onto Sertoli feeder cells, they differentiated into functional sperm that gave rise to offspring. Oxygen at the concentration of air proved to be detrimental for sperm differentiation from SSCs, but not for propagation of SSCs. These results indicate that the whole spermatogenic process can be represented in cell culture in zebrafish, facilitating analyses of the molecular mechanisms of spermatogenesis in vertebrates.

show abstract

“…Furthermore, Langenau et al, 15 have demonstrated that combining the coinjection approach with a heat-shock-inducible transgenic approach can induce transgene expression by heat shock in the primarily injected fish, which would be very useful in exploring the cooperative roles of oncogenes in tumor progression, as it permits gene expression to be turned on after the primary tumor is established. Very recently, Langenau and colleagues developed the first immunocompromised zebrafish model by targeting rag2 gene with engineered zinc-finger nucleases 43 . Loss of function of rag2 permitted robust and long-term engraftment of multiple tissues and cancer cells 43 , an advantage that could be useful in transplanting the heterogeneous cancer cells that overexpress candidate transgenes and assessing the effect of mosaic transgene expression on self-renewal capacity, therapeutic responses and the growth rate of these cancer cells.…”

Section: Discussionmentioning

confidence: 99%

“…Very recently, Langenau and colleagues developed the first immunocompromised zebrafish model by targeting rag2 gene with engineered zinc-finger nucleases 43 . Loss of function of rag2 permitted robust and long-term engraftment of multiple tissues and cancer cells 43 , an advantage that could be useful in transplanting the heterogeneous cancer cells that overexpress candidate transgenes and assessing the effect of mosaic transgene expression on self-renewal capacity, therapeutic responses and the growth rate of these cancer cells.…”

Section: Discussionmentioning

confidence: 99%

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Ung

Guo

Zhu

et al. 2015

JoVE

View full text Add to dashboard Cite

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic "drivers" among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment. Video LinkThe video component of this article can be found at

show abstract

Optimized cell transplantation using adult rag2 mutant zebrafish

Cited by 130 publications

References 24 publications

T Cell Immune Deficiency in zap70 Mutant Zebrafish

T Cell Immune Deficiency in zap70 Mutant Zebrafish

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Contact Info

Product

Resources

About