TSA Activates Pluripotency Factors in Porcine Recloned Embryos

Round 1

Reviewer 1 Report

The work shows interesting results. I found some language mistakes in the article, please double-check the manuscript in this regard. Due to the lack of a line counter, the proposed corrections will be based on sections and paragraphs.

Material and methods: Where were the fibroblasts derived from? Were these fibroblast commercial lines or were they derived for this experiment?

Somatic cell nuclear transfer subsection: there is the wrong font used for "performed"

Establishment of PEFs subsection: the degree symbols are underlined

Quantitative real-time PCR subsection: Is GADPH a properly selected housekeeping gene? Are there any literature data or have tests been carried out?

Bisulfite sequencing subsection: Did you mean "MethylDetector" and "Hot Start"?

Results: Many of the sentences fit more into the discussion section. I propose that sentences covering: the last four and a half lines of the first subsection (Monitoring reprogramming…); the first three and a half lines of the second subsection (Identification of TSA…); a fragment of a sentence in the fourth line of the third subsection (Development enchantment ...) referring to other studies; the last sentence of the same paragraph in this subsection; and the last two sentences of the fourth subsection (RNA expression ...) transfer to the discussion section and create appropriate paragraphs on their basis.

Discussion: The section, in my opinion, does not refer too much to the results obtained in this work and their comparison with the available literature. I suggest you expand it, or combine it with the results section and create a new conclusion section.

Table S1: The table is missing accession numbers for the reference sequences from which the primer sequences were designed. Please provide them. Additionally, the fonts used are not uniform, please improve.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors used OCT4 or SOX2 reporter system to monitor the reprogramming process in pig cloned and re-cloned embryos. They also used three histone deacethyltransferase inhibitors (Trichostatin A, valproic acid and Scriptaid) and found that Trichostatin A could activate pluripotent factors and significantly enhance the development competence of pig re-cloned embryos.  Overall, the results were enough to support the conclusions, especially even they found OCT4/SOX2 reporter genes were up-regulated in TSA-treated groups, the endogenous OCT4/SOX2 were not different. This is an important point to discuss how to improve cloning efficiency in SCNT embryos. However, I’d like to indicate some minor points below.

Abstract
-To over come this issue which occurred frequently in the process of animal recloning, we established…

Introduction
-p.2 L.2 [8-10] -> [8, 9]
-p.2 L.15 [11] -> [10]
-p.2 L.11 [12, 13] -> [11, 12]
-p.2 L.13 [14] -> [13]
-p.2 L.14 [15-18] -> [14-18] Are these correct?

Materials and 
-Please add the permission number of animal experiments.
-p.3 L.18 “performed” -> change the font size

-Results
p.5 L.39-40 (the last sentence of the second section) This seems to be over-interpretation, as only OCT4/SOX2 expression changes were observed at this time. No blastocyst rate, successful rate of cloning were observed (next section). Please re-write this sentence.

-References
7. Metoba -> Matoba
(Please check the reference format again)

Fig.1A pNS-pOCT4-EGFP -> pNP-pOCT4-EGFP?
pNS-pSOX2-tdTomato -> pNP-pSOX2-tdTomato?
Fig.2B pS2-cloned embryos - pST-cloned embryos?

Table S1. “For methylation-specific PCR” -> “For bisulfite PCR” (not methylation-specific PCR)
What do “pOUT4-S” and “pOUT4-A” mean? Why are they not capital? 
 

Author Response

Please see the attachment.

Author Response File: Author Response.docx