Production of mice using iPS cells and tetraploid complementation
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China. Nature Protocol
(Impact Factor: 9.67).
04/2010; 5(5):963-71. DOI: 10.1038/nprot.2010.61
Induced pluripotent stem cells (iPSCs) are considered to be an attractive alternative to embryonic stem cells (ESCs) and may provide great potential for clinical applications in regenerative medicine. Although possessing characteristics similar to ESCs, the true pluripotency of these newly studied iPSCs was not known because none of the previously developed iPSCs passed the tetraploid complementation assay, which is regarded as the most stringent test for pluripotency. We have recently shown that by modifying some of the culture conditions for inducing iPSCs, we were able to generate cell lines of high pluripotency, resulting in the production of live-born, fertile animals through tetraploid complementation. In this paper, we describe details of our methods of generating iPS cell lines and subsequently producing full-term live animals through the tetraploid complementation assay; the procedure can be completed within 2 months.
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Available from: Masakazu Kamata
- "It is now possible to reprogram fully differentiated somatic cells back to the embryonic state by forced expression of certain transcriptional factors such as OCT4, SOX2, C-MYC and KLF4. These reprogrammed cells, termed ‘induced pluripotent stem cells’ (iPSCs), are capable of unlimited self-renewal and display full pluripotency –. The generation of iPSCs offers a new perspective on the use of stem cells in the regenerative medicine field. "
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ABSTRACT: Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.
PLoS ONE 10/2014; 9(10):e108682. DOI:10.1371/journal.pone.0108682 · 3.23 Impact Factor
Available from: Harvey R Herschman
- "Sequencing confirmed Cox2.2058 shRNA in Hygromycin-resistant ESC clones. Transgenic mice were then generated using tetraploid embryo complementation, to give rise to mice that are derived directly from the targeted ES cells –. "
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ABSTRACT: Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3'untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.
PLoS ONE 07/2014; 9(7):e101263. DOI:10.1371/journal.pone.0101263 · 3.23 Impact Factor
Available from: link.springer.com
- "Cell fate transition was shown to be induced or promoted by switching different culture media. A commercially available serum replacer (Knockout Serum Replacer, KSR) has been reported to promote the growth of pluripotent stem cells (Zhao et al., 2010). N2B27 medium was used to isolate rat embryonic stem cells (Buehr et al., 2008) and was successfully used in the establishment of rat naïve ESCs combined with Erk inhibitor PD0325901(PD) and Gsk3 inhibitor CHIR99021(CH) (Li et al., 2008). "
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ABSTRACT: The pluripotent state between human and mouse embryonic stem cells is different. Pluripotent state of human embryonic stem cells (ESCs) is believed to be primed and is similar with that of mouse epiblast stem cells (EpiSCs), which is different from the naïve state of mouse ESCs. Human ESCs could be converted into a naïve state through exogenous expression of defined transcription factors (Hanna et al., 2010). Here we report a rapid conversion of human ESCs to mouse ESC-like naïve states only by modifying the culture conditions. These converted human ESCs, which we called mhESCs (mouse ESC-like human ESCs), have normal karyotype, allow single cell passage, exhibit domed morphology like mouse ESCs and express some pluripotent markers similar with mouse ESCs. Thus the rapid conversion established a naïve pluripotency in human ESCs like mouse ESCs, and provided a new model to study the regulation of pluripotency.
Protein & Cell 01/2012; 3(1):71-9. DOI:10.1007/s13238-012-2007-8 · 3.25 Impact Factor
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