一応丹羽さんと清成さんは１１５４個の胚移植を行って３７１個の発生を
確認したが、キメラ貢献は一つも無かったと報告しているんだね。
>>
Among 1154 embryos injected with the aggregates, 671 developed beyond E8.5;
however, none of the aggregates made significant contribution to any tissues,
the details of which was reported in a Biorixiv website (bioRxiv doi:
*ttp://dx.doi.org/10.1101/028472).
丹羽論文Figure3-aだね。ESはOct4蛋白質の発現量に完全比例してOct4-GFPが発現している。
ところがnon-treatもATPもHCLもOct4蛋白質が全く発現していないのにOct4-GFPが発現している。
この原因は細胞を解離すした時の荒っぽいやり方が原因だということにならざるを得ないね。
丹羽さんはこの発見に関して、Interestinglyと書きだしているが、そんな安易な発見でなかったことには
今頃はお気づきなのかもしれないね。
>>
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.
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-genes[13], 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. 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.
(英文)
Figure 3: Q-PCR analysis for the expression of pluripotency markers in induced cell aggregates.
Figure 3
(a) Q-PCR analysis of the low-pH treated liver cells cultured for 7 days. Liver cells were prepared from 7-day old GOF mice and treated with either ATP or HCl, or without stressor. RNA samples were prepared from all cells in the wells at day 7 of culture and the relative expression levels of Gfp (derived from GOF Tg) and Oct3/4 (derived from the endogenous Pou5f1 allele) to Gapdh were indicated with standard deviation. The expression levels in control ES cells carrying CAG-GFP Tg were set at 1.0. (b) Q-PCR analysis of the single cell aggregates derived from the ATP-treated or non-treated liver cells cultured for seven days. The liver cells were prepared from 4-days old of C57BL6/129 mice and the single cell aggregates were separately treated for quantification of gene expression. The relative expression levels of pluripotency-associated genes to Gnb2l1 were indicated with standard deviation. The expression levels in 10 control ES cells were set at 1.0. (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.
