26 ABERRANT REPROGRAMMING OF IMPRINTED GENE EXPRESSION IN ENLARGED PLACENTAS OF MICE CLONED FROM ES CELLS TREATED WITH TSA OR 5AZAC
Bibliographic record
Abstract
The success rate of producing cloned animals is very low, and in many cases is associated with the formation of enlarged placentas. Increasing evidence has pointed towards epigenetic deregulation of imprinted genes due to incomplete or abnormal resetting of DNA methylation and/or histone acetylation patterns during development. It has previously been shown that drugs that alter DNA methylation (5AzaC) and histone acetylation (TSA) over-express imprinted genes in mouse ES cells (Baqir and Smith, 2001, Theriogenology 55, 410). Our objective in this study was to determine whether nuclear transfer is able to reprogram imprinted gene expression patterns in the placenta of mice cloned from ES donor nuclei exposed to 5AzaC and TSA. ES donor cells were treated with either TSA or 5AzaC prior to injection into enucleated oocytes. Total RNA was extracted from placentas of day 14–15 fetus clones, and reversed transcribed; the expression pattern of imprinted genes (Ipl, Mash2, Igf2, H19, Igf2r, p57, Peg1), non-imprinted placental-specific genes (Esx1, Dlx3, Tpbp) and a housekeeping gene (Gapdh) was examined by Real Time PCR. Samples were standardized with an exogenous control (Globin) and expressed as fold changes in relation to placentas of cloned fetus derived from non-treated donor cells. Data were analyzed by ANOVA and mean gene expression values were compared using the Tukey-Kramer test. Our results show that several imprinted genes (Mash2, H19, Ipl) and placenta-specific genes (Esx1 and Dlx1) were properly reprogrammed in non-enlarged (71 mg) placentas of fetus clones derived from the TSA and 5AzaC treated ES donor cells. Although Gapdh expression did not differ among normal and enlarged 210 mg) placenta groups, the expression level of Igf2 and Mash2 was higher in enlarged placentas from fetus clones produced from TSA-treated ES donor cells (4.6 and 3.5 fold) compared to non-enlarged placentas from non-treated ES cells (1 fold). Conversely, oversized placentas from cloned fetuses derived from TSA-treated donor ES cells under-expressed Peg1, H19 and Ipl (0.5, 0.2 and 0.2 fold, respectively) compared to control placentas (1 fold). In addition, enlarged placentas from the TSA- and 5AzaC-treated group displayed down-regulation of placenta specific genes Esx1 and Dlx3 and up-regulation of Tpbp, suggesting the presence of abnormal distribution of placental layers. These results indicate that while several imprinted and non-imprinted placenta specific genes were correctly expressed in normal size placentas of fetus clones derived from TSA and 5AzaC treated donor ES cells, enlarged placentas displayed aberrant gene expression patterns, suggesting that improper resetting of the epigenetic program after nuclear transfer is directly related to altered DNA methylation and histone acetylation patterns. Funded by NSERC & CIHR.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".