Chromosomal Silencing Induced by Xist Expression from Autosomal Transgenes

Xist transgenes (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes. We investigate an autosomal integration of Xist Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We show ectopic Xist expression in the transgenic male cells along with a transcriptional reduction of genes clustered in two regions of Chromosome 1. RNA-seq and DNA Fluorescent in situ Hybridization (FISH) confirms that Xist Tg is associated with Chromosome 1. TgBAC8 mouse lines were generated and described previously as transgenic mice carrying 200 kb of sequence from the Xic including Xist (Sun et al. 2015). C57BL/6J-TgBAC8-2087 line was used in this study, for which Tg2087/+ mice were crossed to wildtype C57B1/6J mice to obtain offspring that contain littermates of transgenic (Tg2087/+) and wildtype (+/+) animals. Tail tip fibroblasts (TTF) were isolated from tail tip tissues of transgenic and wildtype mice from the same litter and grown in DMEM medium containing 10% FBS supplemented with MEM non-essential amino acids, 25mM HEPES pH 7.2-7.5, 0.1mM 2-mercaptoethanl, penicillin, and streptomycin. TTFs were then immortalized with Simian virus 40 large T cDNA following the protocol as previously described (Brown et al. 1986). Transgenic samples and wildtype (control) samples were processed in parallel following the same procedure for RNA-seq.