Rat Embryonic Stem Cells (rESCs) are used to generate knockout rats (1-3).

1. Transcriptome Profiling of Authentic Rat Embryonic Stem Cells Through RNA-Seq Nathan T. Johnson1, Nripesh Prasad2,3, Shawn E. Levy3, Elizabeth C. Bryda1 1Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 2Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama 3 Hudson Alpha Institute of Biotechnology, Huntsville, Alabama Abstract • Sample Gene Comparison • Embryonic Stem Cell Comparison Gene List Rat Embryonic Stem Cells (rESCs) are a critical tool for producing targeted knockout animals.  However, the transcriptome of rESCs has not been described.   The goals of our study are 1) to characterize the rESC transcriptome and determine what genes are expressed, 2) to better establish an unique expression profile for authentic rESC lines, and 3) to compare the rESC transcriptome with that of human and mouse ESC transcriptomes to gain insight into ESC expression patterns across species.  In order to establish a rESC transcriptome, RNA-seq was done on mRNA from the rESC cell line DAc8, the first male germline competent rESC line to be described and also the first to be used to generate a knockout rat model.  We detected novel isoforms and unannotated rat genes confirmed by RT-PCR and compared and contrasted human, mouse, and rat ESC pluripotency and differentiation genes. Table 1. Gene lists for embryonic stem cell comparison. A. Figure 2. Scatter plot of both rESC samples. Two independent DAc8 samples were analyzed. 23, 281 genes were detected. After stringent filtering by requiring at least 20 reads per gene, a total of 14,358 genes were used for analysis. Four genes had significant expression differences of greater then 1 fold difference between the samples, which was confirmed by Real Time RT-PCR. Novel Isoform Analysis Background • Rat Embryonic Stem Cells (rESCs) are used to generate knockout rats (1-3). • The rESC transcriptome has not been characterized. • RNA-seq is a high throughput sensitive assay, which sequences all RNA in a sample and can be used to establish a transcriptome (4). A. B. Sequencing Strategy B. Figure 3. Example of novel isoform confirmation. 266 total number of novel isoforms were detected at >30% confidence level. Novel isoforms were determined by comparing data output from the ENSEMBL Rat RGSC 3.4 database (March, 2010) and the UCSC RGSC 3.4 database (Feb, 2012). Three novel predicted isoforms were chosen for confirmation by RT-PCR and nucleotide sequence analysis. A) Three known isoforms for Ilf3. Our data predicted 2 novel isoforms due to exon skipping. B) RT-PCR results using primers that flank exons 1-4 (lanes 1-3), exons 1-5 (lanes 4-6), and exons 1-2 (lanes 7-9). Lanes 1,4,7: rESC DAc8 sample, Lanes 2,5,8: rat brain (positive control), Lanes 3,6,9: no template (negative control). A molecular size standard is shown on the left and right . The bands at 15 bp and 1000 bp in all samples are alignment markers. RT-PCR and nucleotide sequence analysis confirmed the presence of the two novel Ilf3_Ratisoforms detected by RNA-seq. A) Comparison of the 10 most highly expressed genes in hESCs, mESCs, and rESCs B) Four (4) genes which were highly expressed in hESCs and mESCs were not detected in rESCs. M= mouse, H= human, R = rat X = expressed Mouse and human data were based on microarray expression analysis (5-7). Summary Exons 2 1 3 4 5 Ilf3 Rat ESCs express novel isoforms of known genes. Gene expression in rESCs is more similar to mESCs than hESCs. As expected pluripotency markers are up-regulated in all 3 types of ESCs. Isoforms • Embryonic Stem Cell Comparison Ilf3_Rat F1LXP4_Rat Ilf3 147 Predicted Isoform References OR 1 2 3 4 5 6 7 8 9 0 63 • Kawamata, M. and T. Ochiya (2011) Cell Mol Life Sci68(11): 1911-1915. • Tong, C., P. Li, et al. (2010) Nature467(7312): 211-213. • Yamamoto, S., M. Nakata, et al. (2011) Transgenic Res. • Mortazavi, A., B. A. Williams, et al. (2008) Nat Methods5(7): 621-628. • Sato, N., I. M. Sanjuan, et al. (2003) Dev Biol260(2): 404-413. • Bhattacharya, B., T. Miura, et al. (2004) Blood103(8): 2956-2964. • Rosenkranz, R., T. Borodina, et al. (2008) Genomics92(4): 187-194. 90 4 91 63 Figure 1. RNA-Seq method used for establishing rESCtranscriptome. Total RNA was extracted from 2 independently maintained cultures of the cell line DAc8. Library prep on total RNA was performed using Illumina’s True Seq to prepare a library by Poly-A selection, fragmentation, and random primer synthesis for each sample. Each library was sequenced with IlluminaHiSeq 2000 using 50 base paired end reads. Output was analyzed using Avadis RNA-seq Pipeline. Acknowledgments: This work is supported by NIH grant P40 RR16939. We thank Howard Wilson for assistance with graphical design Figure 4. Comparison of genes expressed in ESCs from rat, mouse, and human. Using available microarray gene expression data for mouse and human (5-7), the top 1% of genes with highest expression in the embryonic stem cells from each species were compared.