Historical Past Of The PR-171

5 and P0 with 18-gauge needles. RNA from both lenses of each embryo or neonate was isolated by TRIzol purification and processed for hybridization to Affymetrix Mouse Genome 430 2.0 Arrays. Expression values were adjusted by quantile normalization and log transformation with RMAExpress, and data were analyzed with BRB-ArrayTools 3.7.0. ( Fig. S2?and?Fig. S3). Class comparison was used to select genes differentially expressed at a significance level of p?AZD9291 sites. Microarray data presented in YES1 this study has been deposited in the GEO public repository (GSE16533). Both lenses from each embryo or neonate were placed in one microcentrifuge tube and homogenized in TRIzol reagent to obtain total RNA (Life Technologies). Five ��g of total RNA was used to generate cDNA using Superscript III reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed using the BioRad iCycler PCR machine. Each PCR reaction contained 0.5?��l of cDNA template, primers at a concentration of 100?nM, and 1�� of SYBR Green Reaction Mix (BioRad). Reactions were performed in triplicate in a total volume of 25?��l and data were analyzed using the ��Ct method, where GAPDH served as the internal control. Each PCR reaction generated only the expected amplicon as shown by the melting-temperature profiles of the final products and gel electrophoresis. Primer sequences are listed in Fig. S1. We utilized a well-characterized transgenic mouse that expresses cre in lens epithelial and fiber cells to delete a conditional allele of E2f3, either alone or in combination with E2f1 or E2f2, at the earliest stages of lens vesicle formation (cry-cre, also known as MLR10; Fig.?1A) ( Zhao et al., 2004). This analysis showed that the combined deletion of any PR-171 two of the three activator E2Fs does not adversely affect lens epithelial cell proliferation or lens development ( Fig. S5A). To avoid compensation that could result from overlapping functions among E2F members ( Tsai et al., 2008), the entire E2f1-3 subset was deleted. The efficient recombination of the E2f3LoxP allele was confirmed by PCR-genotyping of laser capture microdissected (LCM) lens tissue ( Fig.?1B). Surprisingly, histological examination of E2f1-3 deficient (TKO) lenses revealed no conspicuous change in lens architecture prior to birth ( Fig.?1C).