Reference gene study for forensic body fluid identification
FORENSIC SCIENCE INTERNATIONAL GENETICS SUPPLEMENT SERIES
Authors: Afolabi, O. A.; Roeder, A. D.; Iyengar, A.; Hadi, S.
Abstract
Reference genes are used in forensic body fluid identification studies to normalise data generated during gene expression experiments. The use of reference genes improves the reliability of qRT-PCR. In this study, 10 most common reference genes UCE, TEF, GAPDH, 18S rRNA, ACTB, B2M, B-Actin, OAZ1, RPS 29 and S15 widely used in forensic body fluid identification studies were selected from relevant literature and qPCR efficiency and sensitivity of all the reference genes was tested using SYBR Green detection. Stability was also assayed using samples stored at room temperature for 6 months using Taqman assay probes. All the markers except TEF displayed high sensitivity and were detected down to 25 pg of RNA input. Stability study demonstrates that B2M, ACTB, RPS29, and UCE are ideal markers for normalization in forensic body fluid identification studies. The study confirms that reference genes should be selected only upon adequate validation of their suitability. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility
CELL STEM CELL
Authors: Xu, Huaigeng; Wang, Bo; Ono, Miyuki; Kagita, Akihiro; Fujii, Kaho; Sasakawa, Noriko; Ueda, Tatsuki; Gee, Peter; Nishikawa, Misato; Nomura, Masaki; Kitaoka, Fumiyo; Takahashi, Tomoko; Okita, Keisuke; Yoshida, Yoshinori; Kaneko, Shin; Hotta, Akitsu
Abstract
Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.