丹羽さんはイントロでさりげなく書いてるんだよね。無論多義的な読み方をすればだけどね。
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Full reprograming of somatic cells results in the acquisition of the ability to give rise to an entire organism, or totipotency; this can be achieved by somatic cell nuclear transfer.
丹羽さんの報告書のマテメソにある以下は相沢さんの研究だわよね。
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R26R-H2B-EGFP transgenic mouse (Rosa-GFP) line was generated by Laboratory for Animal Resources and Genetic Engineering (LARGE), RIKEN CDB17.
ここもとてもおかしい場所でね。彼らは<Article のメソッド>だけを言ってるんだが
レターにもこのマウスが出ているよね。例のFigure1のaなんだぜ。ES細胞だ。
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Here we have investigated the unique nature of STAP cells, focusing on their differentiation potential into the two major categories (embryonic and placental lineages) of cells in the blastocyst. We became particularly interested in this question after a blastocyst injection assay revealed an unexpected finding. In general, progeny of injected ES cells are found in the embryonic portion of the chimaera, but rarely in the placental portion (Fig. 1a; shown with Rosa26-GFP). Surprisingly, injected STAP cells contributed not only to the embryo but also to the placenta and fetal membranes (Fig. 1b and Extended Data Fig. 1a–c) in 60% of the chimaeric embryos (Fig. 1c).
アーティクルの部分はこれね。
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For establishment of STAP stem-cell lines, STAP cell clusters were transferred to ACTH-containing medium36 on MEF feeder cells (several clusters, up to a dozen clusters, per well of 96-well plates). Four to seven days later, the cells were subjected to the first passage using a conventional trypsin method, and suspended cells were plated in ES maintain medium containing 20% FBS. Subsequent passaging was performed at a split ratio of 1:10 every second day before they reached subconfluency. We tested the following three different genetic backgrounds of mice for STAP stem-cell establishment from STAP cell clusters, and observed reproducible data of establishment: C57BL/6 carrying Oct4-gfp (29 of 29), 129/Sv carrying Rosa26-gfp (2 of 2) and 129/Sv × C57BL/6 carrying cag-gfp (12 of 16). STAP stem cells with all these genetic backgrounds showed chimaera-forming activity.
うん、そこはど素人なんでよくわからないな。相沢さんのマウスはどこかでてでくるかな?
肝臓で論文通りに近い数が出たんでそこを中心に調べてるな。
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Induced cell aggregates show poor induction of pluripotency-associated markers
To test the induction of pluripotency markers in cell aggregates obtained from ATP-treated liver cells, we assessed the expression of pluripotency-associated genes. Oct3/4 is a well-defined marker of pluripotent stem cells. Using a primer pair to detect Oct3/4 transcript from the Pou5f1 allele, but not pseudo-genes13, we did not find a detectable level (above 0.1% of the expression level in mouse ES cells, relative to the expression levels of Gapdh) of the transcript by quantitative polymerase chain reaction (Q-PCR) using a total RNA sample prepared from all cells in the culture (Fig. 3a), indicating that extremely few or no cells expressing Oct3/4 were present.
こんなことってあるのね。
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Interestingly, expression of Gfp from the Oct3/4-GFP transgene (GOF18)14 was detected in liver cells cultured for seven days irrespective of ATP treatment, suggesting leaky expression of this transgene.
そうだよ。丹羽さんも同じことをしている。
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We next performed qPCR on individual cell aggregates isolated from culture. Aggregates were selected and RNA samples were prepared separately. These RNAs were reverse-transcribed and qPCR was performed.
