RASSF9 Regulated Epidermal Homeostasis Figure 2. Chromosomal disruption and RASSF9 gene silence in transgenic mutant line. (A) Southern blot analysis. Genomic DNA samples from mice of various genotypes were separately digested with HindIII or EcoRI. The left panel showed that a single hybridization band was detected when the transgene was used as the probe. The right panel confirmed a deletion of the identified region in the homozygous mice, as determined by the lack of signal from a probe locating this region. (B) Physical map of the RASSF9 gene. The sequences flanking the inserted transgene in mutant mice were identified by positional cloning. Symbols: white box, RIF; gray box, H3F; black line, intronic DNA; dashed line, the region deleted by the insertional mutagenesis. (C) Total RNA extracted from the indicated tissues of mice of the WT and RASSF92/2 (2/2) genotypes (n = 3 per genotype) were analyzed by RT-PCR using RASSF9 and b-Actin gene-specific primers. b-Actin was used as the internal control for respective samples. The RT-PCR product was analyzed by 2% agarose gel electrophoresis. Similar results were obtained from two independent experiments in panel A. +/+, wild type; +/2, heterozygotes; 2/2; homozygotes. doi:10.1371/journal.pone.0017867.g002 pathology of the transgenic mice may be attributable to abolition of endogenous gene expression rather than the consequence of transgenic LMP-1 expression. Taken together, the results strongly suggest that the transgene insertion in this mutant line unexpect- edly disrupted a gene whose expression was crucial for development. Chromosomal deletion and gene silencing of the RASSF9 gene in the homozygous mutant line In order to elucidate the molecular mechanism of the transgene insertion in the mutant mouse line, we determined the chromosomal integration site of transgene by Southern blot analysis using the transgene as the probe (Tg_probe; Supple- mentary Information, Table S1). A single band was detected upon restriction digestion of the genomic DNA by the enzymes HindIII and EcoRI, suggesting that the transgene had integrated at a single locus, as the transgene contained no restriction sites targeted by the enzymes (Figure 2A, left panel). In order to identify the chromosomal location of the transgene-insertion site, we used an inverse PCR approach [15]. Two flanking fragments (designated H3F and RIF; Figure 2B) containing short unique DNA sequences that were not part of the transgene were recovered. The flanking DNA fragments, which were assigned to the mouse chromosome 10D1 region, were separated by about 7.2 kb, indicating that integration of the transgene resulted in the deletion of about 7.2 kb of the genome at the insertion site (Figure 2B). To confirm these results, we used the same blot to perform Southern hybridization with a probe located within the putative 7.2-kb deletion (D_probe; Table S1); no signal was detected in homozygous mutant (2/2) mice (Figure 2A, right panel). The deleted genomic sequence was mapped to an intron sequence approximately 178 bp downstream of the first exon of the gene encoding RASSF9 (Figure 2B). The mapping of the deletion/transgenic insertion to murine RASSF9 gene (accession number: AK041688) was confirmed with the genomic sequence from the latest assembly of the UCSC Genome Browser [July 2007 assembly (NCBI37/ mm9); http://genome.ucsc.edu]. Thus, we concluded that the transgenic mice harbored a 7.2-kb chromosomal deletion within the 32.2-kb intron of the RASSF9 gene, in close proximity to its first exon. In this study, we routinely performed genotype screening by genomic PCR using primers targeting the LMP-1 transgene and the deleted sequence of the RASSF9 intron (Figure S2A, bottom two panels). Further analyses of tissue samples from homozygotes (2/2) demonstrated that the chromosomal deletion resulted in RASSF9 gene silencing in PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17867