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Integrating viral vectors*

SAFETY. EFFICIENCY. Integrating viral vectors*. Excisable viral vectors*. Reprogramming proteins. piggyBac transposon. Non-integrating vectors. Eliminates risks associated with genome modification; non-DNA approach Very slow process.

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Integrating viral vectors*

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  1. SAFETY EFFICIENCY Integrating viral vectors* Excisable viral vectors* Reprogramming proteins piggyBac transposon Non-integrating vectors Eliminates risks associated with genome modification; non-DNA approach Very slow process Precise transposon excision leaves no ‘footprints’ of exogenous DNA Time-consuming; excision may be inefficient Low frequency of integration Repeated transfection/infection required; possibility of random insertion of vector fragments Easy to use; reproducible; allows secondary iPSC generation Increased risk of insertional mutagenesis; possibility of transgene reactivation; incomplete silencing (lentiviruses) Efficient Cre-mediated removal of transgenes Exogenous DNA (primarily viral LTR) remains integrated following excision advantages disadvantages REPROGRAMMING Differentiated somatic cell iPSCs REPROGRAMMING FACTORS MOUSE: Oct4 (Nr5a2), Klf4 (Esrrb, Klf2 or Klf5), Sox2 (Sox1), c-Myc (N-Myc or L-Myc) HUMAN: Oct4, Klf4, Sox2 , c-Myc, Nanog, Lin28, hTERT, SV40LT SMALL MOLECULES# VPA (histone deacetylase inhibitor); BIX-01294 (histone methyltransferase inhibitor); vitamin C; 5’-azaC (DNA methyltransferase inhibitor); BIO (GSK3 inhibitor); ALK5 and RepSox (inhibitors of TGF-β signaling); BayK8644 (L-type Calcium channel agonist); kenpaullone (kinase inhibitor) STARTING CELL POPULATION MOUSE: embryonic and adult fibroblasts, neural stem cells, hepatocytes, gastric epithelial cells, B- cells HUMAN: keratinocytes, skin fibroblasts, embryonic fibroblasts, CD34+ peripheral blood cells Cell types with high endogenous levels of a reprogramming factor require a minimal cocktail of factors Small molecules can substitute for a reprogramming factor and/or enhance reprogramming efficiency

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