Preparing Recombinant Gonad Organ Cultures

It can be useful to assay migration between any two adjacent tissues during development. This protocol assays cell migration between the gonad and mesonephros using tissue recombination between genetically marked and unmarked tissue, combined with an organ culture technique. First, agar blocks are prepared in a custom-built mold. The size and shape of the wells are important to maintain the authentic three-dimensional morphology of the organ; the molds here are designed specifically to accommodate the gonad/mesonephros complex. Freshly dissected organs are then transferred to grooves within the agar blocks, where they are allowed to grow over 24-48 h. Using this protocol, organs develop with good morphology, and show only an ~12-h delay relative to in vivo development.