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FIGURE 5 Localized PUV treatment and heat-shock apparatus to label cells in the Xenopus cornea. (aâc) Localized PUV-treatment procedure.(a) MS-222 anesthetized tadpole is placed in a petri dish on Kimwipes soaked in anesthetic solution. Reservoir pipette tip is affixed to a metal rodheld by the micromanipulator using a small binder clip. The tip is gently placed around the cornea to form a sealed chamber. Psoralen solution isadded into the reservoir and allowed to contact the cornea. (b) Higher magnification view of area shown in panel (a). (c) Image through the 10Ãobjective showing aluminum foil covering the anterior half of the cornea, restricting UV illumination to the posterior half. Aluminum foil was firstplaced on top of a glass coverslip, which was then placed on the moistened cornea to avoid drying out of the tissue (We found that drying of thecornea can activate some expression in these cells). (d) Transgenic animals contain a transgenic construct that uses the hsp70 promoter for heat-shock inducible H2B-mCherry expression in nuclei and GFP-GPI expression in cell membranes. The second portion of the construct contains agamma-crystallin promoter driving GFP expression in the lenses of these transgenic animals. The transgenic animals carrying this construct werecreated by the sperm nuclear injection method of transgenesis (Smith et al., 2006), which were then reared to sexual maturity and mated toobtain F1 transgenic animals to be utilized for these experiments (see Appendix 1 for details). The sexually mature transgenic frogs have beendeposited to the National Xenopus Resource (NXR), and transgenic tadpoles carrying this construct can be obtained from the NXR. (e,e0)Convenient sorting of the transgenic versus nontransgenic animals is made possible by observing the presence vs. absence of the GFP expressinglenses, respectively. (f) Heat-shock probe to induce localized expression in a specific cluster of cells. The probe is attached to a micromanipulatorand the sharpened nichrome filament is gently touched to the desired location on the cornea. (g) Complete set-up of the heat-shock apparatusshowing various components, as indicated. Tadpoles at Stages 51â53 (Nieuwkoop & Faber, 1994) immobilized in a clay lined petridish containing1/20Ã Normal Amphibian Media, after anesthetizing, as described by Adil et al. (2019). Electric current is applied to the filament to achieve thedesired temperature of 35â37îC for 6 min. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC)and the Division of Animal Resources (DAR) at the University of Illinois. Images in (a,b) are reprinted from Adil, M. T., Simons, C. M., Sonam, S., &Henry, J. J. Understanding cornea homeostasis and wound healing using a novel model of stem cell deficiency in Xenopus, Experimental EyeResearch, 187, 107767, Copyright (2019), with permission from Elsevier. Image in (d) is based on Hamilton and Henry (2014), Prolonged in vivoimaging of Xenopus laevis, Henry, J. J. & Hamilton, P. W., published in Developmental Dynamics by John Wiley and Sons
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