アーティクルのTCRの件は以下ね。
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TCR-β chain gene rearrangement analysis
Genomic DNA was extracted from STAP cells and tail tips from chimaeric mice generated with STAP cells derived from CD45+ cells. PCR was performed with 50 ng DNA using the following primers (Dβ2: 5′-GCACCTGTGGGGAAGAAACT-3′ and Jβ2.6: 5′-TGAGAGCTGTCTCCTACTATCGATT-3′) that amplify the regions of the (D)J recombination. The PCR products were subjected to gel electrophoresis in Tris-acetate-EDTA buffer with 1.6% agarose and visualized by staining with ethidium bromide. PCR bands from STAP cells were subjected to sequencing analysis and identified as rearranged genomic fragments of the (D)J recombination.
我々は小保方さんは無実という立場だからね。当然だけどB6-CAG-GFPx129/Svだと
若山さんに言われたからそう書いていて、かつ親の雌雄の違いは見た目では分からないよね。
そして「あるとき」ではなくて2012年の3月に光る胎盤を伴うキメラ胎児を
受け取って、それを若山さんの指示通りに、切片の免染確認でGFPを胎盤に検出しているのが
Letter Extended Data Figure 1 Placental contribution of STAP cellsなんだよな。
*ttps://ipscell.com/2014/02/are-stap-stem-cell-nature-papers-compromised/
Nimorula
February 25, 2014 at 7:19 pm .
In the paper “Bidirectional developmental potential…” besides the duplicated image in figures 1 and 2 there are things really hard to believe about the data they show in figure 1. First they should have done the analysis in chimaeric embryos previous to the chrorio-allantoic fusion (embryos before 6 somites, around E8) to conclude that only STAPs and not ES cells contribute to the placenta. Developing my point: After the allantois (derived from the embryonic mesoderm) fuses with the chorion (extraembryonic) the formation of the labyrinthine placenta occurs. In this, the trophoblast (extraembryonic) associates with the fetal blood vessels (embryonic) that have extensive villous branching. Then in an E12.5 placenta from a chimaera formed from a wildtype blastocyst injected with wildtype reporter Rosa26-GFP or CAG-GFP ES cells you should always find GFP positive cells in the placenta due to the contribution of the fetal blood vessels. Then is not possible that in the control chimaeras in figure 1a they didn’t find Rosa26-GFP cells (as stated in the text) / CAG-GFP (as stated in the figure). The same applies for the fetal membranes, the yolk sac mesoderm is derived from the embryo then in a chimaera you always have ES-derived cells (mesoderm) in the yolk sac. There is no way that control is real. Mild: the long exposure panel of the control embryo was not overexposed since the little piece of embryo in the far right of the panel is not overexposed.
nimorula
February 26, 2014 at 2:21 pm .
p.s. A nice example of chimaeric embryos analysing the contribution of ES cell derived tissue to the placenta in E12.5 embryos can be found in PNAS USA 100(26):15637-15642. (2003) see figure 4.
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『Failure to replicate the STAP cell phenomenon』
An anomalous allele frequency distribution observed in FI-SCs, and reciprocal analyses of FI-SC heterozygous SNVs in TSCs and TSC homozygous and heterozygous SNVs in FI-SCs, revealed that FI-SCs were derived from a C57BL6 strain origin, with approximately 10% contamination from TSCs (Fig. 1i and Extended Data Fig. 3). These are concordant with the findings from a recent RIKEN report .
㉖本論に戻ってDBの＜C57BL/6x129/Sv＞としたのは、小保方さんなのか、ラボ仲間の手伝いをしてくれた人なのか、
登録時の理研の事務員なのかを明らかにしろ。小保方さんは親マウスの雌雄をちゃんとルールに従って記載している。
②129/Sv x B6-CAG-GFP
③B6-CAG-GFP x 129/Sv
は書き分けられている。
論文にはSTAP幹細胞転換培養のところで、使われたストレーンのＦ1は②だと明確に書かれている。
まずは論文は以下だ。
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STAP stem-cell conversion culture
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.
何度も言うが、<論文/DB>/DBという分類はまやかしだ。二つは別々のもので、
まずSTAPに関して由来マウスは
①GOFマウス
②129/Sv x B6-CAG-GFP
③B6-CAG-GFP x 129/Sv
④B6-CAG-GFP x DBA/2
⑤129/Sv-Rosa26-GFP
が記載されているだけだ。他に使われたものはあるかも知れないがそれは記載がない。
