Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.
Part I. Set-up for the ACT Assay
Part II. RNA Microinjection of Xenopus laevis embryos
A. Preparation for microinjection
B. Microinjection
Part III. Isolate animal caps from injected embryos
Part IV. Transplantation of caps onto flank
Figure 1. Design of Plexiglass for casting the elastomer mold used for making embryo holding dishes. A 38 x 38 mm round-cornered square (~5 mm deep) has been routed out of a piece of Plexiglass that is 50 x 50 mm square and 25 mm thick. Circular, evenly spaced wells (1.5 mm diameter) have been drilled 1 mm deep into the plastic. To make the elastomer mold for the microinjection dishes, we poured Sylgard elastomer into the Plexiglass and allowed it to solidify, as per manufacturer instructions. This elastomer mold is used to make injection and cap isolation plates.
Figure 2. Animal cap sorting prior to transplantation is important, as expression can vary. Animal cap on the left expresses mCherry evenly throughout tissue while the explant on the right has spotty expression and will be discarded.
Figure 3. A host tadpole has developed an eye-like structure on its flank from transplanted animal cap cells. A fluorescent image has been overlayed on the brightfield image.
In this video, we have demonstrated our version of classic techniques used by Xenopus biologists, which we rearranged to create the animal cap transplant assay. At room temperature, Xenopus embryos develop very quickly through stages 9 and 15. By placing them at 14°C, the development of the embryo is slowed down and many more transplants can be performed in one experiment. If it is necessary to grow the embryos to older stages (>42/43), then we recommend using transgenic venus YFP animals, since the injected fluorescent protein signal fades in older tadpoles. We used this assay to establish that animal cap tissue expressing EFTFs or Noggin is specified to form eye-like tissue. This assay could be used as a simple test to establish whether a gene(s) of interest is necessary for determining a particular cell fate.
This work was supported by grants from Research to Prevent Blindness (Career Development Awards to MEZ and ASV and an unrestricted grant to the Department of Ophthalmology), the E. Matilda Zeigler Foundation (MEZ and ASV) and the National Eye Institute/NIH (MEZ, grants R01EY015748 and R01EY017964).
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
mMessage mMachine SP6 Kit | Applied Biosystems/Ambion | AM1340 | ||
Borosilicate Capillary Tubes (1.0mm OD x 0.5mm ID) | FHC, Inc | 27-30-1 | ||
Sutter Micropipette Puller | Sutter Instruments, Inc. | P-97 | ||
Gentamicin sulfate [50 mg/ml] | Fisher | BW17-528Z | ||
Sylgard/elastomer Kit 1.1lb | Fisher | NC9644388 | ||
60 x 15 mm polystyrene petri dish | USA Scientific | 8609-0160 | ||
human chorionic gonadotropin | Intervet | 22219 | ||
Pico-Injector Microinjection Systems | Harvard Apparatus | 650003 | ||
Torrey Pines Scientific ECHOtherm Incubator | Fisher | 11-680-21 | ||
Transfer pipette | Krackeler | 6290-20635A | ||
Dumostar #5 Forceps | Fisher | 11295-10 |