Transplantation of Whole Kidney Marrow in Adult Zebrafish

Summary

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Abstract

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (<3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

Protocol

Irradiation

1. Irradiate WT fish at 25Gy 2 days before transplantation

Kidney dissection

1. Sacrifice appropriate number of fluorescent donors in tricaine (~100ml 4mg/ml Tricaine / liter of fish water) (average of 500,000 cells per donor fish)
2. Isolate kidney into 0.9X PBS + 5%FCS + 1%Penn strep ("PBS+") in tube
3. Break up with 1ml pipet, then with 10ml syringe with 18 1/2 G needle
4. Transfer into 50mL conical and fill up with PBS+
5. Spin down @ 1500rpm 8min
6. Pour out, resuspend pellet in large volume and filter through 40micron filter
7. Spin down @ 1500rpm 8min
8. Pour ....