A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

The overall goal of this method is to locate and track the migration, expansion and survival of stem cells and progenitor cells in the corneal epithelium. This method can help answer key questions in the corneal epithelial cell biology field concerning homeostasis, tissue repair, aging, and Pathology. The main advantage of this technique is that it is easy to establish and it allows non-invasive lineage tracing of cells in a clonal manner.

To begin, first select a Cree transgenic mouse strain with an inducible tissue specific promoter to label the limbal and corneal epithelial progenitor cells and stem cells. The Tamoxifen Inducible K 14 Cree ERT line is used. Next, choose the appropriate brainbow cassette depending on the research question and the available Cree line.

In this case, the Brainbow 2.1 line is used. Cross the homozygous R 26 R confetti strain with the homozygous K 14 Cree ERT strain to generate mice with four color K 14 positive cells To induce recombination in the transgenic mice. First weigh 80 milligrams of Tamoxifen and dissolve it in one milliliter of corn oil by Vortexing.

Place the solution in a shaking water bath, maintained at 65 degrees Celsius until it completely dissolves, and leave it in the bath until use next anesthetize. An eight week old Brainbow 2.1 Cree ERT heterozygous mouse, and check the depth of anesthesia with the toe pinch carefully apply 100 microliters of the Tamoxifen solution to each ocular surface of the mouse. Direct application of Tamoxifen yields a high efficiency of Cree induction Store any residual Tamoxifen solution protected from the light at four degrees Celsius for up to one week.

Warm the solution prior to application, which is repeated on three consecutive days for each mouse. First dissolve one gram of Tamoxifen powder in five milliliters of sterile dimethyl sulfoxide. To produce a 200 milligram per milliliter solution, set the solution in a shaking water bath, maintained its 65 degrees Celsius until it becomes clear and leave it in the bath until use.

Then anesthetize the mouse and check for depth of anesthesia using a toe pinch, apply eye ointment to the untreated eye of the mouse to protect it from dehydration during the procedure and administer adequate analgesics. Next, draw 200 microliters of the Tamoxifen solution into a syringe without any needle attached, and apply it topically over the entire cornea of the eye. The liquid solidifies on the tissue at room temperature.

Attend to the mouse until it maintains sternal recumbent and return it to the company of others only when it is fully conscious. After sacrificing mice at appropriate time points post induction, an eye wounding as described in the text protocol. Nucleate the eye by first pressing the eyelid to pop the eyeball out of the socket.

Slide curved forceps behind the eyeball to hold the optic nerve and pull the eyeball up to detach it. Place each eyeball in a 60 millimeter dish containing PBS. Place the eye containing plate under a surgical stereo microscope.

Make sure you recognize the basic structure of the eye, the cornea, limbus, and the optic nerve. Remove the optic nerve, muscle, and connective tissue around the eye. Hold the eye at the posterior part with the cornea facing down, and make an incision in the sclera at the back of the eyeball using spring scissors.

Enlarge the cut to allow the lens to pop out and remove the iris with forceps. Next, make four to five radial incisions by cutting from the center towards the periphery to flatten the cornea and then remove any remaining portions of the iris with a thin flat spatula. Finally, cut the excess peripheral tissue surrounding the limbus to fix the cornea.

Transfer the dissected tissue to a 12 well dish and incubate it in 4%para of formaldehyde for 10 minutes. Then briefly wash the tissue in PBS to stain the cell nuclei. Incubate the cornea in two micrograms per milliliter of DPI solution for 10 minutes, followed by a brief wash in PBS.

Next, place the cornea on a glass slide with the epithelial side facing up. Then place a drop of mounting media on top of the cornea and cover it with a cover slip. Let the slides dry overnight at room temperature and store them at four degrees Celsius.

For corneal imaging, use a spectral confocal system that enables separation of R-F-P-Y-F-P-G-F-P and CFP emissions. First set the fluorescence, excitation, and emission wavelengths depending on the fluorescent proteins of the brain. Bo cassette, taking care to prevent cross excitation or overlap of emission signals.

Next, acquire tiled images to visualize the entire cornea in high resolution. Here, obtain an array of 12 by 12 images in all four fluorescent channels. Finally, merge the images to create a composite lineage.

Tracing experiments using R 26 R confetti mice were performed to follow the fate of limbal and corneal epithelial progenitor cells. Under steady state conditions, small clusters of fluorescent cells were observed 10 days after induction as expected. After four months, streaks from the limbus to the center of the cornea developed suggesting that cells migrate from the limbus to regenerate the cornea following corneal injury.

Saw migration from the limbus occurred rapidly with long, thick limbal stripes developing within seven days. This model is valuable for tracking and studying corneal stem and progenitor cells under different circumstances. It'll allow the investigation of clonal cell expansion, survival, and migration under steady state and under stress conditions such as wounding, chemical burn mutation, and more In the future.

This model may also be used to dissect the molecular mechanisms underlying normal and pathological corneal regeneration. This technique may contribute to our understanding of different pathological conditions such as corneal dystrophies, burns, and corneal trauma, and in addition to the evaluation of the impact of different treatments on corneal regeneration, cell expansion, and migration.