Immune Compromises Zebrafish for Cell Transplantation

描述（由申请人提供）：将细胞移植到免疫受损小鼠中改变了我们对人类疾病的理解，并已广泛用于评估再生、干细胞自我更新和癌症。尽管异种移植细胞移植到免疫受损小鼠中的巨大效用，但小鼠模型由于高饲养成本而不适于大规模研究，并且不便于移植细胞的直接可视化。B相反，斑马鱼价格便宜，可以大量饲养，并且适合大规模的化学遗传方法，其中化合物可以直接添加到水中。光学清晰的卡斯帕-品系斑马鱼缺乏色素沉着，便于直接观察 活体成年动物体内的荧光细胞。此外，利用基因相同的同基因斑马鱼品系进行细胞移植已经证实，使用数百只受体动物进行大规模细胞移植是可能的。最后，斑马鱼可以在35 ℃下饲养，因此是人类和小鼠细胞移植的潜在模型。尽管斑马鱼模型具有这些明显的优势，但使用免疫受损的斑马鱼进行异种移植细胞移植尚未报道。利用光学透明的卡斯帕品系斑马鱼， 最近开发的使用TALEN的基因失活，我们将开发具有在免疫受损小鼠中发现的类似突变的斑马鱼系，其通常用于异种移植细胞移植。目的1将开发叉头框Nl（foxnl/nude）、重组激活基因2（rag 2）、Janus激酶3（jak 3）、DNA依赖性蛋白激酶（prkdc）和白细胞介素2受体γ（IL 2 rg）缺陷的斑马鱼品系。目标2将评估每种模型植入荧光标记的斑马鱼、小鼠和人的正常和恶性细胞的能力。将在单突变体鱼和复合突变体中评估移植物植入：1）rag 2-/-、jak 3-/-; 2）rag 2-/-、112 rga-/-、112 rgb-/-; 3）prkdc-/-、jak 3-/-;和4）prkc-/-、IL 2 rga-/-、Il 2 rgb-/-。这些实验将允许全面比较斑马鱼突变系之间的植入。目标3将建立一个组织结构，以快速有效地将可行的突变株系分发给研究界。这里概述的实验将为社区开发急需的资源，促进下一代低成本，高通量细胞移植模型。免疫受损斑马鱼的这种广泛应用跨越了许多NIH研究所的使命。

DESCRIPTION (provided by applicant): Cell transplantation into immune compromised mice has transformed our understanding of human disease and has been used extensively to assess regeneration, stem cell self-renewal, and cancer. Despite the great utility of xenograft cell transplantation into immune compromised mice, mouse models are not amenable to large-scale studies due to high husbandry cost and do not facilitate direct visualization of engrafted cells. B contrast, zebrafish are inexpensive, can be reared in large numbers, and are amenable to large-scale chemical genetic approaches where compounds can be added directly to the water. Optically clear caspar-strain zebrafish lack pigmentation, facilitating the direct visualization of fluorescent cells in live adult animals. Moreover, cell transplantation utilizing genetically-identical, syngeneic zebrafish strains has validated that large-scale cell transplantation using hundreds of recipient animals is possible. Finally, zebrafish can be raised at 35C and thus a potential model for engraftment of human and mouse cells. Despite these clear advantages of the zebrafish model, the use of immune compromised zebrafish for xenograft cell transplantation has yet to be reported. Capitalizing on optically-clear caspar-strain zebrafish and recently developed gene inactivation using TALENs, we will develop zebrafish lines with similar mutations found in immune compromised mice that are commonly employed for xenograft cell transplantation. Aim 1 will develop zebrafish lines that are deficient in forkhead box N l (foxnl/nude), recombination-activating gene 2 (rag2), Janus kinase 3 (jak3), DNA-dependent protein kinase (prkdc), and interieukin 2-receptor gamma (IL2rg). Aim 2 will assess each model for the ability to engraft fluorescent-labeled normal and malignant cells from zebrafish, mouse, and human. Transplant engraftment will be assessed in single mutant fish and compound mutants for 1) rag2-/-, jak3-/-; 2) rag2-/-, 112rga-/-, 112rgb-/-; 3) prkdc-/-, jak3-/-; and 4) prkc-/-, IL2rga-/-, Il2rgb-/-. These experiments will allow comprehensive comparison of engraftment between zebrafish mutant lines. Aim 3 will establish an organizational structure to rapidly and efficiently distribute viable mutant lines to the research community. The experiments outlined here will develop a much-needed resource for the community, facilitating the next generation of low-cost, high throughput cell transplantation models. Such broad reaching applications for immune compromised zebrafish spans the mission of many NIH institutes.