A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models

Abstract

Patient-derived induced pluripotent stem cells (iPSCs) are valuable tools for the study of developmental biology and disease modeling. In both applications, genetic correction of patient iPSCs is a powerful method to understand the specific contribution of a gene(s) in development or diseased state(s). Here, we describe a protocol for the targeted integration of a doxycycline-inducible transgene expression system in a safe harbor site in iPSCs. Our gene targeting strategy uses zinc finger nucleases (ZFNs) to enhance homologous recombination at the AAVS1 safe harbor locus, thus increasing the efficiency of the site-specific integration of the two targeting vectors that make up the doxycycline-inducible system. Importantly, the use of dual-drug selection in our system increases the efficiency of positive selection for double-targeted clones to >50 %, permitting a less laborious screening process. If desired, this protocol can also be adapted to allow the use of tissue-specific promoters to drive gene expression instead of the doxycycline-inducible promoter (TRE). Additionally, this protocol is also compatible with the use of Transcription-Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system in place of ZFNs.

DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells

“…Furthermore, deletion of the enhancer occupied by SMAD3 was also associated with a significant reduction in mRNA and protein expression of SOX17, FOXA2 and CXCR4 (Figures 1I, 1J, S1G and S1H), which together identify DE (D’Amour et al, 2005; Green et al, 2011; Loh et al, 2014; Ogawa et al, 2013). Passage-matched hESCs and hESCs containing a green fluorescent protein (GFP) expression system (Sim et al, 2015) were used as controls. We used the CRISPR system to insert the components of the GFP expression system into the AAVS1 loci to create a control that had undergone the same manipulations used to create the SMAD3 enhancer deletions.…”

Understanding platelet generation from megakaryocytes: implications for in vitro–derived platelets

“…36 Inducible and megakaryocyte transgene expression can also be performed using the PPP1R12C (AAVS1) "safe harbor" locus, which prevents deleterious off-target cloning and gene silencing that can occur during differentiation. [69][70][71][72] Modulation of transcriptional master regulators, such as PU.1, can be used to eliminate unwanted myeloid cell fates 71 that may contribute to the enzymatic injuries of megakaryocytes.…”

LiPS-A3S, a human genomic site for robust expression of inserted transgenes

“…It was originally recovered as a natural site of integration of wild-type AAV in cultured human cell lines 5 and gained popularity due to, on one hand, its ability to afford robust expression of inserted reporter transgenes in various cell types, including hPSCs (embryonic stem cells and induced pluripotent stem cells (iPSCs)) and, on the other hand, the development of efficient commercially available tools for its targeting. 6,7,8,9 However, more systematic recent studies have revealed that the AAVS1 locus is prone to silencing in some cell lineages. 10 Furthermore, a number of applications require dual transgene or reporter expression or transgene insertion in hPSC lines where the AAVS1 locus is already occupied by other transgenic sequences, for example by an inducible genome editing enzyme.…”