Abstract
We describe a procedure for generating induced pluripotent stem cell lines in rabbits, using retroviral vectors expressing Oct4, Sox2, Klf4, and c-Myc of human origin to reprogram rabbit fibroblasts prepared from an ear skin biopsy. We also provide detailed procedures for characterizing the resulting iPSC lines, including the analysis of pluripotency marker expression by RT-qPCR, immunolabeling, and fluorescent-associated cell sorting, the evaluation of pluripotency by teratoma production and genetic stability by karyotyping.
Keywords:
Induced pluripotent stem cells; Klf4; Oct4; Pluripotency genes; Rabbit; Reprogramming; Retroviral vectors; Skin fibroblasts; Sox2; c-Myc.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Culture Techniques / methods
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Cell Differentiation
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Cells, Cultured
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Cellular Reprogramming
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Cellular Reprogramming Techniques / methods
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Chimera / genetics
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Ear, External / cytology
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Fibroblasts / cytology
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Gene Expression
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Genetic Vectors / genetics
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Humans
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Induced Pluripotent Stem Cells / cytology*
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Induced Pluripotent Stem Cells / transplantation
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Karyotyping
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Kruppel-Like Factor 4
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Kruppel-Like Transcription Factors / genetics
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Kruppel-Like Transcription Factors / physiology
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Mice, SCID
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Morula / cytology
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Octamer Transcription Factor-3 / genetics
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Octamer Transcription Factor-3 / physiology
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Proto-Oncogene Proteins c-myc / genetics
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Proto-Oncogene Proteins c-myc / physiology
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Rabbits*
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Recombinant Proteins / genetics
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Retroviridae / genetics
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SOXB1 Transcription Factors / genetics
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SOXB1 Transcription Factors / physiology
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Teratoma / pathology
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Transgenes
Substances
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KLF4 protein, human
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Klf4 protein, mouse
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Kruppel-Like Factor 4
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Kruppel-Like Transcription Factors
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MYC protein, human
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Octamer Transcription Factor-3
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POU5F1 protein, human
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Proto-Oncogene Proteins c-myc
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Recombinant Proteins
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SOX2 protein, human
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SOXB1 Transcription Factors