Transgenic mice with defined combinations of drug-inducible reprogramming factors (original) (raw)
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Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts. 1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types. 2,3 Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine. 4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells. Protocol
A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types
Nature Biotechnology, 2008
The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25-to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.
2010
Here we demonstrate that the doxycycline-inducible expression of the mouse transcription factors Oct4, Klf4, Sox2, and c-Myc contained within a single 4F2A polycistronic mRNA is sufficient to reprogram Oct4-neo MEFs to iPS cells when the 4F2A polycistronic lentivirus is co-transduced with a reverse tetracycline transcriptional activator lentivirus. These VSV-G pseudotyped lentiviruses are capable of infecting both dividing and non-dividing cells from any mammalian species. Additionally, we show that G418 is useful for iPS cell colony selection for expansion as the Oct4-neo MEFs contain a neomycin resistance gene targeted to the endogenous Oct4 locus. Endogenous expression of the neomycin resistance gene from the Oct4 locus is a clear indication that the iPS cell colonies are indeed pluripotent and capable of self-renewal. Lastly, we demonstrate that the iPS cell colonies generated display typical ES cell-like morphology and express common mouse ES cell pluripotency markers such as S...
Development of an All-in-One Inducible Lentiviral Vector for Gene Specific Analysis of Reprogramming
PLoS ONE, 2012
Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.
A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency
Cell Stem Cell, 2008
Current approaches to reprogram human somatic cells to pluripotent iPS cells utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous "secondary" somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes. These cells were obtained by infecting fibroblasts with DOX-inducible lentiviruses, isolating "primary" iPS cells in the presence of the drug, and finally differentiating to "secondary" fibroblasts. When "secondary" fibroblast lines were cultured in the presence of DOX without further viral infection, up to 2% of the cells were reprogrammed to pluripotent "secondary" human iPS cells. This system will facilitate the characterization of the reprogramming process and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors.
Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic Cells
Cell Stem Cell, 2008
Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4 and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox) inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, were only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step towards understanding some of the molecular events governing epigenetic reprogramming.