In this video, we demonstrate how to express exogenous DNA constructs in optic neurons and how to image individual GFP-expressing optic axonal arbors in intact, living Xenopus laevis tadpoles. This is an inexpensive, simple procedure for transient cell-specific transgenesis that allows determination of cell autonomous gene function in individual optic axonal arbors, developing in a living vertebrate model system. Demonstrating the procedure will be myself, Sophia Dao, Research Assistant, and Dr.Tamira Elul, Lab Principal Investigator.
To begin, gently clip the tip of a pulled glass micro capillary pipette with fine forceps. Backfill the glass micro capillary pipette with mineral oil using a MICROFIL such that a tiny drop of mineral oil appears at the clipped tip of the micro pipette. Fill the glass micro capillary pipette halfway with mineral oil.
In an injector, eject the plunger halfway and load the pulled glass micro capillary pipette into the injection holder. Then extend the plunger to the full extent to confirm that the micro capillary pipette is strongly attached to the injector and does not move with the extension of the plunger. Transfer a three microliter drop of the DNA/DOTAP mixture onto a one inch square sheet of paraffin paper.
Under a stereo dissecting microscope, move the tip of the glass micro capillary pipette into the DNA/DOTAP drop. Use the fill option on the injection apparatus, slowly suck the DNA/DOTAP drop into the glass micro capillary pipette. The boundary between the mineral oil and the DNA/DOTAP solution is visible in the glass micro capillary pipette due to the slight opacity of the DNA/DOTAP solution.
If needed, stop filling the micro capillary pipette periodically to allow the pressure in the glass micro capillary pipette to recalibrate. First, in a 10 milliliter Petri dish filled with 0.1X MMR, manually de-vitellinize 10 stage 20 to 24 Xenopus embryos with fine forceps. Grasp the vitelline envelope at the waist to avoid injuring the embryos.
With forceps in both the experimenter's left and right hand, pop the bubble of the vitelline envelope and release the embryo from the vitelline envelope. Take care not to injure the embryos when removing the vitelline envelope. Then use a plastic transfer pipette with a cut tip to transfer five to 10 de-vitellinized stage 22 to 24 embryos to a 10 milliliter Petri dish filled with 1X MMR.
Under a stereo microscope, grasp one of the de-vitellinized embryos in the Petri dish with forceps and arrange the embryo so that its anterior pole is pointed up in the field of view. Then orient the embryo so that it is lying laterally and one of its left to right eye buds is facing upwards. Hold the embryo with the forceps in the experimenter's nondominant hand and with the experimenter's dominant hand, introduce the tip of the glass micro pipette from the ventral or dorsal side just beneath the epidermis into the eye bud.
Inject between 70 to 210 nanoliters of the DNA/DOTAP solution. Then turn the embryo around and perform the same micro injection into the other eye bud on the contralateral side of the embryo. Inject both eye buds of six to 10 embryos in each experiment.
After micro injection, store embryos in a Petri dish with 1X MMR for approximately 30 minutes to facilitate wound healing. After 30 minutes, transfer the injected embryos with a plastic transfer pipette into a 0.1X MMR solution with 0.001%bleaching agent phenylthiocarbamide to reduce pigmentation. Cover the Petri dish with a lid to culture the embryos for approximately five days until the embryos have developed into tadpoles at stages 46 to 47.
This protocol yields a success rate of 30 to 60%of injected Xenopus embryos expressing GFP in one to 10 optic axonal arbors. Representative confocal images of GFP show the expressing control and mutant optic axonal arbors in the intact Xenopus tadpoles. Two domain mutants of APC, APCNTERM, and APBbeta-cat were cloned into pCS2 plasmids.
Z-series images of GFP control and APC mutant optic axonal arbors were reconstructed. Plots of number of branches, total arbor branch length, and mean branch length confirm observed differences between control and APC mutant expressing axonal arbors. Additional scatter plots of number of branches versus mean branch length with regression lines show inverse correlation between these parameters and optic axonal arbors expressing APC domains.
The tip of the micro capillary pipette must be correctly inserted very superficially into the eye bud so that the gray epidermis overlying the eye bud swells during each micro injection. This procedure can be used to both label and alter specific genetic function in optic neurons while assessing the growing, targeting, and branching of axons from these optic neurons. This DNA micro injection and lipofection technique has allowed researchers in developmental neurobiology to study cell autonomous molecular mechanisms that regulate optic axon arborization in intact, living Xenopus laevis tadpoles.
