Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study, we describe the use of a gammaretroviral vector encoding a fluorescent marker, mRFP1, to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming

Design and Assembly of CRISPR/Cas9 Lentiviral and rAAV Vectors for Targeted Genome Editing

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system has emerged as an extremely useful tool for biological research and as a potential technology for gene therapy approaches. CRISPR/Cas mediated genome editing can be used to easily and efficiently modify endogenous genes in a large variety of cells and organisms. Furthermore, a modified version of the Cas9 nuclease has been developed that can be used for regulation of endogenous gene expression and labeling of genomic loci, among other applications. This chapter provides an introduction to the basis of the technology and a detail protocol for the most classic application: gene inactivation by CRISPR/Cas9 nuclease system from Streptococcus pyogenes. This workflow can be easily adapted for other CRISPR systems and applications

Combined preconditioning and in vivo chemoselection with 6-thioguanine alone achieves highly efficient reconstitution of normal hematopoiesis with HPRT-deficient bone marrow

Purine analogs such as 6-thioguanine (6TG) cause myelotoxicity upon conversion into nucleotides by hypoxanthine-guanine phosphoribosyltransferase (HPRT). Here we have developed a novel and highly efficient strategy employing 6TG as a single agent for both conditioning and in vivo chemoselection of HPRT-deficient HSC. The dose-response and time course of 6TG myelotoxicity were first compared in HPRT-wild type mice and HPRT-deficient transgenic mice. Dosage and schedule parameters were optimized to employ 6TG for myelo-suppressive conditioning, immediately followed by in vivo chemoselection of HPRT-deficient transgenic donor bone marrow (BM) transplanted into syngeneic HPRT-wild type recipients. At appropriate doses, 6TG induced selective myelotoxicity without any adverse effects on extra-hematopoietic tissues in HPRT-wild type mice, while HSC deficient in HPRT activity were highly resistant to its cytotoxic effects. Combined 6TG conditioning and post transplant chemoselection consistently achieved ~95% engraftment of HPRT-deficient donor BM, with low overall toxicity. Long-term reconstitution of immunophenotypically normal BM was achieved in both primary and secondary recipients. Our results provide proof-of-concept that single-agent 6TG can be used both for myelo-suppressive conditioning without requiring irradiation, and for in vivo chemoselection of HPRT-deficient donor cells. Our results show that by applying the myelosuppressive effects of 6TG both before (as conditioning) and after transplantation (as chemoselection), highly efficient engraftment of HPRT-deficient hematopoietic stem cells can be achieved