RASSF9 Regulated Epidermal Homeostasis Figure 1. Characterization of the transgenic mutant mouse line. (A) Macroscopic phenotypes of mice of various genotypes were shown at one, two, and six weeks after birth. (B) Growth curve analysis. The growth curves of the different genotypes exhibited an effect of haploinsufficiency on body weight. The body weight of the homozygous mutant was severely reduced as compared to that of the heterozygous and WT mice. (C) The survival rate. The survival rate of transgenic mutant mice was lower than that of the WT mice. Statistical analyses were performed using the SPSS13.0 software. P,0.05. +/+, wild type; +/2, heterozygous mutant; 2/2, homozygous mutant. doi:10.1371/journal.pone.0017867.g001 12,0006 g for 10 minutes at 4uC. Total RNA was precipitated from the resultant aqueous phase by isopropanol, washed in 70% v/v ethanol, air-dried and then resuspended in RNAse-free water for subsequent assays. The A260/A280 ratio of all RNA samples was between 1.8 and 2.1, and sample quality was verified by electrophoresis on 1% agarose gel for 18S and 28S ribosomal RNAs. The resultant samples of total RNA were submitted to the Microarray & Gene Expression Analysis Core Facility, VGH National Yang-Ming University Genome Research Center, Taipei, Taiwan, for subsequent processing and assay with Affymetrix GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Fremont, CA). All the data is MIAME compliant, and the raw datasets are deposited in MIAME-compliant NCBI-Gene Expres- sion Omnibus (GEO) database (Accession Number: GSE24190). The resulting dataset was analyzed using GeneSpring software (Silicon Genetics, Agilent Technologies, Santa Clara, CA). Image quantitation and statistical analysis Quantitation of image data was achieved using the image- analysis programs ImageJ. (version 1.41) and ImageQuant (version 5.1). Analysis for statistical significance was examined using t-tests. P values ,0.05 were considered statistically significant. Results Characteristics of a transgene-insertion-mutation mouse line Out of 21 independent Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transgenic mice, one mouse line exhibited intriguing phenotypes showing growth retardation, alopecia and short lifespan in homozygotes (2/2; Figure 1A– C), and signs of haploinsufficiency for heterozygotes (+/2) for the alopecia (Figure 1A). Homozygous mice (2/2) were born with normal Mendelian frequency but started to die off rapidly after two weeks past birth. At six weeks past birth, approximately 10% of homozygous mutants survived to wean (Figure 1C, left panel), but no remaining homozygotes survived beyond one year after weaning. Overall, homozygous exhibited severe growth retarda- tion and short lifespan compared to heterozygous and the WT mice (Figure 1A–C). The mRNA transcript of LMP-1 transgene in this line was not efficiently expressed in the neonate skin at the onset of the pathology (Figure S2A, top panel). Meanwhile, LMP-1 protein expression in the skin of the transgenic mice at two weeks of age was not detectable by Western blotting with anti-LMP-1 antibody (Figure S2B). Thus, it is likely that the observed skin PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17867