Finally, we demonstrated that Lin 28 is sufficient to induce RPE transdifferentiation in chick reti nectomized eyes in the absence of exogenous FGF2. The conservation of a dedifferentiation molecular profile be tween regenerative models including retina, lens and limb or fin regeneration is indicative of a common process to reprogram selleck chem cells to a plastic state, where the cells can be directed to expand and respond to environmental cues in order to differentiate and replace lost cells and tissues. Methods Chick embryos and surgical procedures Fertilized Specific Pathogen Free chicken eggs were incu bated in a humidified rotating incubator at 38 C. At E4, retinectomies and FGF2 treatments were performed as previously described. Embryos were collected at 6, 24 and 72 h PR and processed for laser capture microdissection, histology and immunofluor escence.
For proliferation studies, 10 ul of BrdU solution was dropped over the eye of the embryo 1 h before collection. Laser capture microdissection Laser capture microdissection was performed as previ ously described. Briefly, embryos were collected and infiltrated at 4 C with 6. 25%, 12. 5% and 25% sucrose for 10, 20 or 30 min, respectively, followed by 2,1 25% sucrose to optimal cutting temperature compound for 1 h and frozen in dry ice and methylbutane. Cryosections were collected onto metal framed polyethylene naphthalate membrane slides, fixed in 70% ethanol at 20 C for 30 s, rinsed in cold diethylpyrocarbonate treated water, stained with hematoxylin for 10 s, and de hydrated in ethanol for 30 s each in 70%, 95% and finally 2 min in 100% ethanol.
Laser capture microdissection was performed using a Veritas laser capture microdissection sys tem and software as described previously. Laser micro dissected sections were collected in CapSure HS LCM Caps, and total RNA extraction was performed using PicoPure RNA Isolation Kit including a treatment with DNase I. The quality and quantity of RNA were deter mined using an Agilent RNA 6000 Pico LabChip. Five nanograms of total RNA with an RNA integrity number 8 were amplified using Ovation Pico WTA System V2 according to the manu facturers instructions, to generate the Single Primer Isother mal Amplification cDNA. Finally, SPIA cDNA was purified using QIAquick PCR Purification Kit and quantified using a Nanodrop ND 100 spectrophotometer.
RT PCR Total RNA was extracted from Stage 8 whole embryos using NucleoSpin RNA II isolation Kit following the manufactures protocol. The quality and quantity of RNA were determined using Agilent selleckchem Calcitriol RNA Nano LabChip. Approximately 300 ng of total RNA with a an RNA integrity number 8 were used for cDNA synthesis using ImProm II Reverse Transcription System and random primer hexamers according to the manufacturers instructions. For CM and RPE, the amplified SPIA cDNA was used as a template in the PCR reactions.