アーティクルは以下ね。
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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.
プロトコルは以下ね。
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(iii) We have established multiple STAP stem cell lines from STAP cells derived from CD45+ haematopoietic cells. Of eight clones examined, none contained the rearranged TCR allele, suggesting the possibility of negative cell-type-dependent bias (including maturation of the cell of origin) for STAP cells to give rise to STAP stem cells in the conversion process. This may be relevant to the fact that STAP cell conversion was less efficient when non-neonatal cells were used as somatic cells of origin in the current protocol.
疑義の意味がなあ?
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tt
@ts_marker
9月30日
その他
" Few of these (22 of 391 cell aggregates) gave rise to colonies of small stem cell-like cells, and one could be passaged three times (Fig. 7b). "
その時の細胞は調べられた？
簡単に言うとそういうことです。小保方さんの初期研究ではスフィア塊20個から
50個に一つ検出されている。丹羽さんの検証実験では50万個の細胞から30個くらいまでの
スフィア塊ができて、そのうちの20%というから6個のスフィア塊にOct4遺伝子の発現が
あった。つまり5個のスフィア塊に1つあったんだから、小保方さんよりとても多く見つけている。
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However, the frequency was very low; 5 × 105 liver cells yielded only ~30 cell aggregates, in which about 20% of the cell aggregates contained 1–2 Oct3/4 positive cells, indicating a frequency per seeded liver cell of 0.0012–0.0024%.
まあ、実際にはcに書かれているように幾分かでも定性PCRでESと比較できそうなくらい
Oct4GFPを発現している27個のスフィア塊をES10個分の量に対してどれだけ
発現しているかをしめしたのがbだと書いてあって、27個中更にES10個分の
0.001以上発現しているスフィア塊だけを選んで14並べたのがb表だと書かれているわね。
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(c) Frequency of cell aggregates showing the levels of Oct3/4 expression comparable to ES cells. The relative expression levels of Oct3/4 in single cell aggregates derived from liver cells were measured as b and the frequency of the cell aggregates with the levels of Oct3/4 expression over 0.001 of relative expression to ES cells is indicated.
