title: Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA.
abstract: The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells.
authors.affiliation: Division of Pediatric Hematology Oncology, Children's Hospital Boston, and Dana-Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yuin-Han
authors.initials: YH
authors.lastname: Loh
authors.forename: Jimmy Chen
authors.initials: JC
authors.lastname: Yang
authors.forename: Alejandro
authors.initials: A
authors.lastname: De Los Angeles
authors.forename: Chunguang
authors.initials: C
authors.lastname: Guo
authors.forename: Anne
authors.initials: A
authors.lastname: Cherry
authors.forename: Derrick J
authors.initials: DJ
authors.lastname: Rossi
authors.forename: In-Hyun
authors.initials: IH
authors.lastname: Park
authors.forename: George Q
authors.initials: GQ
authors.lastname: Daley
mesh_terms: D000818:Animals; D065150:Cellular Reprogramming / Q000235:genetics*; D061252:Feeder Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D019426:Integrases / Q000235:genetics*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D016254:Mutagenesis, Insertional / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D012333:RNA, Messenger; C045073:Cre recombinase; D019426:Integrases