129B6F1G1 and GR14 are nuclear transfer-
derived ES(ntES) cell lines (Wakayama et al
2001) previously established in our laboratory
using sertoli cell from a 129B6F1 background
With GFP(Ohta ＆ Wakayama 2005) and
tail tip cell of a mail mous
(129BDF2. Wakayama et al 2005) respectively
as donor for nuclear transfer karyotype
analysis revealed that the ES cell lines
Used had the normal karyotype at the
following percentages (number of metaphases
with normal karyotype in parentheses)
46%(12／26)for E14 35%(9／26) for
129B6F1G1 and54%(14／26)for GR14
①2005年論文(これが太田さんのntESを作った時の実験。ただし、Lさんのおっしゃる通り、この論文自体はクローンの実験。)
Generation of Normal Progeny by Intracytoplasmic Sperm Injection Following Grafting of Testicular Tissue from Cloned Mice That Died Postnatally
Hiroshi Ohta Teruhiko Wakayama
Biology of Reproduction, Volume 73, Issue 3, 1 September 2005, Pages 390–395, doi.org/10.1095/biolreprod.105.041673
Published: 01 September 2005
②2008年8月論文(坂出さんとの共著論文)
Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages
Hiroshi Ohta, Yuko Sakaide and Teruhiko Wakayama
Received 30 April 2008
Revision requested 28 May 2008
Revision received 15 July 2008
Accepted 28 August 2008This Article
Published online before print August 29, 2008, doi: 10.1530/REP-08-0184
③2008年9月論文(特許関連論文)
Increasing the Cell Number of Host Tetraploid Embryos Can Improve the Production of Mice Derived from Embryonic Stem Cells
Hiroshi Ohta Yuko Sakaide Kazuo Yamagata Teruhiko Wakayama
Biology of Reproduction, Volume 79, Issue 3, 1 September 2008, Pages 486–492, doi.org/10.1095/biolreprod.107.067116
Published: 01 September 2008
この論文のマテメソに、以下のようにあるわ。
>>
The 129B6F1G1 [13] and BDmt2 [14] are nuclear transfer-derived ES (ntES) cell lines [15] previously established in our laboratory using Sertoli cells of 129B6F1 background with GFP and tail-tip cells of a male BDF1 mouse as donor for nuclear transfer, respectively. Male 129B6F1 mice expressing GFP were generated by mating female 129/Sv mice with male C57BL/6 GFP transgenic mice (double-transgenic mouse line [16] constructed using pCAG-EGFP [10, 11] and Acr3-EGFP [17]).
あなたが引用したのは②の論文の以下ね。
>>
129B6F1G1 and GR14 are nuclear transfer-derived ES (ntES) cell lines (Wakayama et al. 2001) previously established in our laboratory using Sertoli cells from a 129B6F1 background with GFP (Ohta & Wakayama 2005) and tail tip cells of a male mouse (129BDF2; Wakayama et al. 2005) respectively, as donors for nuclear transfer. Karyotype analysis revealed that the ES cell lines used had the normal karyotype at the following percentages (number of metaphases with normal karyotype in parentheses): 46% (12/26) for E14; 35% (9/26) for 129B6F1G1; and 54% (14/26) for GR14.
ここで<論文に書かれていたのはクレアの129+Ter>だと言ってるのは2005年論文のことね。
マテメソに書かれている。Terだわね。
>>
To generate 129B6F1 mice carrying the GFP transgene, female 129/Sv-ter mice were mated with male C57BL/6 GFP transgenic mice, and the offspring of these matings, which were hemizygous for the GFP transgene, were used as donors for nuclear transplantation.
それはむしろ2008年論文のために作成されたntESじゃないかな。というのも
例えば2008/8月論文には履歴があるよね。
Received 30 April 2008
Revision requested 28 May 2008
Revision received 15 July 2008
Accepted 28 August 2008
論文が提出されたのは4/30でアクセプトされたのが8/28だ。4か月かかっている。
論文のための実験はそれ以前に行われているんだから2007/8/7凍結というのは
論文提出10か月前だよ。小保方さんの実験なんて2年がかりなんだからね。この
G1が特許関連論文のみかぃとだというのは時間的な不整合はないね。
彼女はヴァカンティ研究室で逆転写PCR実験によってOct4蛋白を発見してから、
ヴァカンティに認められ、その後ラボの全員の協力を得てあのティシュー誌に
載せられている各マーカー蛋白のゲル上の電気泳動バンドの図表を作ったんだね。
>>
Although the spheres in this study described were capable of generating teratoma-like tissue, the transplanted cells did not form large teratomas as did ES cells, nor they did express Eras in vitro as ESCs,31 which suggests that the teratoma-like tissue they generate may be very different from true teratomas generated from ESCs. In addition, the cells studied did not express the trophectoderm marker Cdx232 or also associated with ESCs. These differences of gene expression pattern may explain the differences of the biological function between ESCs and adult stem cells in this study.
レター論文の参照は以下ね。
>>
16.
Yang, J. et al. Stat3 activation is limiting for reprogramming to ground state pluripotency. Cell Stem Cell 7, 319–328 (2010)
Show context
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PubMed
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ど素人には手が回らないね。あはははは。
>>
However, as Fgf4-induced stem cells lay between STAP stem cells and trophoblast stem cells in the dendrogram, the possibility of contamination of STAP stem cells in the Fgf4-induced stem-cell population cannot be ruled out. Previous studies have indicated that inner cell mass (ICM)-type pluripotent cells can be removed from culture by treating the culture with a JAK inhibitor <16>(Extended Data Fig. 5a, b). In contrast, the JAK inhibitor treatment had no substantial effect on Oct4-GFP expression in Fgf4-induced stem-cell culture (Extended Data Fig. 5c, d; see Extended Data Fig. 5e, f for control). Expression of neither pluripotency markers (Fig. 2j) nor trophoblast markers (Fig. 2k) was substantially affected, indicating that pluripotency marker expression is unlikely to reflect contaminating STAP stem cells (ICM-type). Consistent with this idea, Fgf4-induced stem cells that were strongly positive for the trophoblast marker Itga7 (a surface marker for trophoblasts but not ES cells) also expressed high levels of Oct4-GFP (Extended Data Fig. 5g).
レター論文のa JAK inhibitor <16>の参照がヤンさんの論文だけど今ざっと
目を通した限りでJAK阻害剤処置でESが取り除かれるとは書かれてないように見える。
にも拘わらず、Extended Data Fig. 5にはその実証証明がある。ヤンさんの論文は
今見つけたばかりだからもうちょっと詳しく調べないと分からないね。
ヤンさんの論文の小保方さんが言ってる部分は以下だわね。
>>
Results
Lif Increases the Efficiency of EpiSC Reprogramming
We compared the frequency of Epi-iPSC generation in the presence or absence of Lif. We used embryo-derived Oct4-GFP (O4G) reporter EpiSCs stably transfected with expression constructs for Klf4 or Nanog (Guo et al., 2009). On transfer from activin plus Fgf into 2i with Lif, these cells produced GFP-positive iPSC colonies at a frequency of 0.5%–1% (Guo et al., 2009, Silva et al., 2009). Without Lif or in the presence of a Jak inhibitor, this yield was reduced several fold (Figure 1A ). To test whether the effect of Lif is due simply to increased efficiency of iPSC self-renewal, we plated reprogrammed Epi-iPSCs in 2i with or without Lif. We observed no significant difference in numbers of colonies formed or their undifferentiated phenotype (Figure 1B).
<Without Lif or in the presence of a Jak inhibitor, this yield was
reduced several fold (Figure 1A ). >のところね。図の1-Aは棒グラフだけど
小保方さんがLetter Extendes Data5で示した写真と同じ結果ね。
もちろんだよ、ワトソン。
>>
However, as Fgf4-induced stem cells lay between STAP stem cells and trophoblast stem cells in the dendrogram, the possibility of contamination of STAP stem cells in the Fgf4-induced stem-cell population cannot be ruled out.
<南北首脳：戦争終結へ、完全な非核化目指す－歴史的な「板門店宣言」>
Bloomberg
2018/04/28 00:56
<核のない朝鮮半島を実現するという共通の目標>なんて認めてないんでね。
目標ではない。即時撤去しろと命令している。無論米軍が乗り込んで確認する。
当たり前だろ。
原文にこうある。 Kanga Kongが工作員だな。
By Kanga Kong and Andy Sharp
2018年4月27日 17:58 JST Updated on 2018年4月27日 21:57 JST
はい、どうぞ。
>>
a, Culture of Oct4-GFP Fgf4-induced cells in LIF + 20% FBS medium. b, qPCR analysis of ES-like cells derived from Fgf4-induced cells for pluripotent marker genes (left) and trophoblast marker genes (right). Values are shown as ratio to the expression level in ES cells (left) or trophoblast stem (TS) cells (right). c, d, Culture of Oct4-GFP Fgf4-induced cells sorted by FACS for strong integrin α7 (Itga7) expression in LIF + 20% FBS medium. d, Formation frequency (shown by percentage) of Oct4-GFP+ colonies from cells plated on gelatin-coated dishes at a clonal density. **P < 0.01; t-test; n = 3. e, f, Culture of Oct4-GFP Fgf4-induced cells (dissociated) in LIF + 20% FBS medium with MEK inhibitor. **P < 0.01; NS, not significant; Tukey’s test; n = 3.
続きよ。
>>
e, No substantial formation of Oct4-GFP+ colonies was seen from Fgf4-induced cells in the presence of MEK inhibitor (left), whereas colonies frequently formed when cells were co-plated with Oct4-GFP ES cells (right; plated cells were 1/20 of Fgf4-induced cells). f, Quantification of colony formation per plated cells (1 × 103 Fgf4-induced cells and/or 1 × 103 ES cells). Unlike Fgf4-induced cells, ES cells formed colonies (regardless of co-plating with FI-SCs) in the presence of MEK inhibitor. Bars and error bars represent mean values and s.d., respectively (b, d, f). Scale bars: 100 μm (a, c, e).
ここだわ。
>>
To confirm further that Fgf4-induced stem cells with a trophoblast-like nature were converted into ES-like cells, rather than just selecting ES-like cells pre-existing in the Fgf4-induced stem cell culture, we examined the effect of the MEK inhibitor PD0325901 on the ES-like cell generation from Fgf4-induced stem cells. Like trophoblast stem cells, Fgf4-induced stem-cell survival is dependent on FGF–MEK signals, and the inhibition of MEK activity caused massive cell death (Extended Data Fig. 6c). However, PD0325901 is also known to be a main effector in 2i medium[17] and to promote ES cell maintenance. Addition of PD0325901 to LIF+FBS-containing medium strongly inhibited the formation of ES-like colonies from Fgf4-induced stem cells (Fig. 3e, left, and Fig. 3f). This inhibition was unlikely to be due to secondary toxic effects from massive cell death of Fgf4-induced stem cells, as colonies formed in the presence of PD0325901 when ES cells were co-plated in the same culture with Fgf4-induced stem cells (Fig. 3e, right, and Fig. 3f).
自明というのはどこまで深く考えたかということとの相関なんだね。
>>
There are more things in heaven and earth, Horatio,than are dreamt of in your philosophy.
桂報告は自明だと言った。which are dreamt of in his philosophy という意味でね。
そしてこのフィロソフィーを持っていた人は桂調査に対して科学的検証結果とその判断を
書いて提出し、桂委員会はその言を採用し、おそらくその文言も借用した。科学検証チームは
複数人でリーダーは今野さんだったかな。でも桂調査委員会に提出した報告書のを書いた人は
もう一段上位の責任者だ。
Ooboeさん、JAKiの実験はレター論文のFigure2-j,kとExtended Data Figure5だわ。
レター論文は
*ttp://www.nature.com/articles/nature12969
で見れる。
本文と本文図表は今有料になって見れないけど、Extended Data Figure5は下の方に
下がっていくとあるからクリックして開いてみるとしいいわ。
<小保方氏はSTAP細胞を作製する際に若山氏から渡され たマウスの遺伝的背景を
把握していなかった>ら、論文書けないでしょと訝ったのね。だったら他の可能性を
考えるものよね。
>>
There are more things in heaven and earth, Horatio,than are dreamt of in your philosophy.
