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田鼠117

银虫 (小有名气)

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专业: 乳腺肿瘤

[交流] 审稿意见回来了,我是否该放弃呢?

谢谢大家了,意见很多,有些还是关于实验的,可惜我现在不可能再补做实验了,所以大家看我是否该放弃呢?谢谢
Reviewer #1:
1) The hematopoietic chimerism (engraftment level of human hematopoiesis) of transplanted mice is not reported.
这句话是什么意思,要我补数据吗?
2) Figure 1B, the AAV proteins do not appear to have the expected ration of 1:1:10. A short comment on this issue would be appreciated.
这个我可以解释

3) Figure 2&3: With the notable exception of Figure 3A, in most experiment a clear difference between transduced and negative control samples cannot be seen. This makes the interpretation of successful gene transfer difficult.

4) Figures 2&3: Can the RT-PCR primers distinguish between AAV DNA and RNA?

5)  To show a proof of transduction, additional PCRs would be helpful to detect AAV backbone DNA (not RNA) sequences, if possible using quantitative or semi-quantitative approaches.

6) The levels of correction noted at the level of protein expression appear to be minor. This should be discussed and approaches to reach therapeutically meaningful levels of correction should be considered.

7) The Discussion largely repeats the thoughts presented in the introduction and results sections and somehow fails to discuss the work in the context of the literature, such as state of the art of thalassemia correction using lentiviral vectors.

Reviewer #2: Major issues:
1. rAAV titers reported here are low.  Other labs routinely make one or two orders of magnitude more concentrated vectors. In Figure 1, what is the number of plasmid molecules for each dilution of the dot-blot?  At the vg/ml concentration reported here, it is very difficult to detect the rAAV capsid protein bands on SDS-PAGE gels even when 5-fold concentrated, so purity evaluation is difficult.  In Figure 1, how were the AAV capsid proteins in the SDS-PAGE gel detected, since the figure looks like a Western blot, not protein staining?

2.The infectious titer of the rAAV preps were not determined, therefore MOI is incorrectly used since MOI is the number of infectious rAAV units per cell.  Change this to "vector genomes per cell" throughout the manuscript, figures, and tables.

3. Details about the culture conditions during the 2 hour ex vivo rAAV transduction are missing.
4. How was separation or removal of single-stranded rAAV DNA from the RNA preparation accomplished prior to RT-PCR analysis?  How do the authors know they are detecting RNA expressed from the vector?
5. What are the timepoints used in Figure 4?  Is Figure 4Ac from transduced mice?  Why weren't untreated NOD/SCID mice included in the Mass Spec analysis (Figure 4B)?

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