Analysis of Dip2B Expression in Adult Mouse Tissues Using the LacZ Reporter Gene
, Noor Bahadar 1,2,†, Fatoumata Binta Bah 1, Salah Adlat 1, Zin Mar Oo 1, Luqing Zhang 1, Fawad Ali 3, M S Zobaer 4, Xuechao Feng 1,* and Yaowu Zheng 1,*Round 1
Reviewer 1 Report
The manuscript is well written and figures are very clear.
Could you add few details on how fluorescent "frozen section images" were processed? Could you add few words on why the fluorescent images of the protein are blue-colored?
Could you add a comment of the possible effect on histological analysis for replacing the gene of interest with LacZ?
The mammalian sequence of Dip2b could be added in the SI, as a reference for readers.
The manuscript lacks references in few places:
In addition to reference #16 could you add few more recent references of LacZ staining imaging applications?
For the following sentence please add a reference if known: "Cytological investigations are ongoing 306 to identify cell type-specific distribution of Dip2b gene expression and to elucidate possible functions of Dip2b gene in these organs."
Could you please refer to the following studies and verify the known function of Dip2b? Please, include these references or as appropriate.
Gerdin AK (2010). "The Sanger Mouse Genetics Programme: High throughput characterisation of knockout mice". Acta Ophthalmologica. 88: 925–7. 10.1111/j.1755-3768.2010.4142.x
van der Weyden L, White JK, Adams DJ, Logan DW (2011). "The mouse genetics toolkit: revealing function and mechanism". Genome Biol. 12 (6): 224. 10.1186/gb-2011-12-6-224
The SI file is unavailable and therefore I couldn't inspect the transcription report or any other content.
Author Response
Comment 1: Could you add few details on how fluorescent "frozen section images" were processed? Could you add few words on why the fluorescent images of the protein are blue-colored?
Responses: Frozen section images were taken by light microscopy because a dark blue precipitate produced after X-gal staining can be easily detected by light microscopes. Fluorescent microscope was not used in this project. We have added couple of sentences in the introduction section.
Line 76-80
Comment 2: Could you add a comment of the possible effect on histological analysis for replacing the gene of interest with LacZ?
Responses: As per our knowledge, there will not be any change in histological analysis for replacing the gene of interest with LacZ. LacZ is a reporter gene, representing promoter activity of gene of interest. It cannot tell the half-life of mRNA of interest, translational efficiencies of mRNA and protein level of gene of interest. Histology analysis of the interested protein would be best. But harder to do without a good antibody.
Comment 3: The mammalian sequence of Dip2b could be added in the SI, as a reference for readers.
Response: Thank you for the valuable suggestion sir, we have now added a Dip2b mice genomic sequence extracted from NCBI in S1 file
Comment 4: The manuscript lacks references in few places:
In addition to reference #16 could you add few more recent references of LacZ staining imaging applications?
Response: Thank you for the comments. We have now added some more references of LacZ staining imaging application.
References 18-24
Comment 5: For the following sentence please add a reference if known: "Cytological investigations are ongoing 306 to identify cell type-specific distribution of Dip2b gene expression and to elucidate possible functions of Dip2b gene in these organs."
Response: We are very thankful for revising intensively, we have now included two references based upon the recent publication.
References 16 and 25
Comment 6: Could you please refer to the following studies and verify the known function of Dip2b? Please, include these references or as appropriate.
Gerdin AK (2010). "The Sanger Mouse Genetics Programme: High throughput characterisation of knockout mice". Acta Ophthalmologica. 88: 925–7. 10.1111/j.1755-3768.2010.4142.x
van der Weyden L, White JK, Adams DJ, Logan DW (2011). "The mouse genetics toolkit: revealing function and mechanism". Genome Biol. 12 (6): 224. 10.1186/gb-2011-12-6-224
Response: Thank you so much for your suggestion. We have now added some recent known function of Dip2b gene based upon the references you have provided.
Comment 7: The SI file is unavailable and therefore I could not inspect the transcription report or any other content.
Response: we are so sorry to know about it. Supplementary information has two files,
File S1. Dip2b disco interacting protein 2 homolog B [Mus musculus (house mouse)] genomic sequence
Figure S2: Whole mount LacZ staining of adult brain.
Reviewer 2 Report
This paper shows the tissue distribution of Dip2B in adult mice using LacZ reporter gene, They showed novel findings, and presented them well; however, revision and modification is required to be suitable for publication.
Major points:
- The authors mentioned quantitative Real Time PCR; however, the results did not show any. In discussion they mentioned figure 12; but it is missing. Please add the results of real time PCR or remove it from methods section/or make the explanation more clear.
- In results section (1. Generation of Dip2b mice) is misleading, did the authors generated these mice, or they purchased them (as mentioned in the methods section), please clarify and give details of your contribution if you participated in the generation process. This subsection of results - as expected from the results shown - is related to confirmation of mice genotype rather than the generation of the mice.
- In Figure 2: the figure caption does not correspond to the image labeling, there are two images labeled the same for each of D, E and F.
- I suggest summarizing the expression results in a table, to make it easier to track the comparison.
- Did the author have biological duplicates for these experiments, please provide the details and the number of mice for each experiment.
- In discussion, More emphasis on the importance and applications of this paper results is required.
Minor points:
- There is a huge number of abbreviations used in the manuscript, please mention the full term of each one of them in the first occurrence and prepare a complete list for all of them.
- Please be consistent when you use gene symbols (italic or not).
Author Response
Major points:
Comment 1: The authors mentioned quantitative Real Time PCR; however, the results did not show any. In discussion they mentioned figure 12; but it is missing. Please add the results of real time PCR or remove it from methods section/or make the explanation clearer.
Response: Thank you so much for pointing out the mistakes. It is an error from our side. Figure 12 was actually included in supplementary information and as it was a supplementary information, we did not include it in a result section. We have now corrected it and decided to add it in a main result as Figure 9.
Comment 2: In results section (1. Generation of Dip2b mice) is misleading, did the authors generated these mice, or they purchased them (as mentioned in the methods section), please clarify and give details of your contribution if you participated in the generation process. This subsection of results - as expected from the results shown - is related to confirmation of mice genotype rather than the generation of the mice.
Response: Reviewer is correct. We have now changed the sentence from Generation of Dip2b mice to Genotyping of Dip2btm1a mice.
Comment 3: In Figure 2: the figure caption does not correspond to the image labeling, there are two images labeled the same for each of D, E and F.
Response: Thank you so much for revising carefully. We have now modified figure 2 and labelled carefully.
Comment 4: I suggest summarizing the expression results in a table, to make it easier to track the comparison.
Response: Thank you so much for your suggestion. We have now included a table (Table 1) listing all the expression results.
Comment 4: Did the author have biological duplicates for these experiments, please provide the details and the number of mice for each experiment.
Response: Yes, we use three biological duplicates for each experiment. We have added more information in animal sub-section under material and method section.
Line 97-98
Comment 5: In discussion, more emphasis on the importance and applications of this paper results is required.
Response: Thank you for your suggestion. Based upon our knowledge, we have discussed our results to a possible extent. We are still not sure which part need to be modified. If the reviewer has any special point needed to be mentioned than please let us know.
Minor points:
Comment 1: There is a huge number of abbreviations used in the manuscript, please mention the full term of each one of them in the first occurrence and prepare a complete list for all of them.
Responses: Thank you so much for reviewing carefully, we have now rechecked every abbreviation.
Comment 2: Please be consistent when you use gene symbols (italic or not).
Response: we have revised it thoroughly and corrected it.
Reviewer 3 Report
Dip2B Expression Analysis in Adult tissues Using LacZ Reporter Gene
In this manuscript, Sah et al. determine dip2b expression in a variety of mouse tissues via reporter gene and reverse transcription PCR. Knowing where dip2b is expressed can then be used in later studies to determine function in knockout animals/cells.
Specific Comments:
Line 128: Please provide primer sequences (or kit manufacturer) for 18S ribosomal RNA detection
Figure 2: The N-P panels (WT) mentioned in the text and figure legend is missing from this figure.
Line 271: There is no Figure 12.
Line 276: Dip2b protein levels were not determined in this study (no antibodies to Dip2b are mentioned). This should be removed from the statement.
Line 282-283: Please clarify this sentence about what the study found (if Dip2b is solely expressed, or if it was knocked out and function was looked at).
Line 301-302: Expression in certain tissues does not suggest a role in cell fate, only that the gene is expressed and should be removed.
Author Response
Comment 1: Line 128: Please provide primer sequences (or kit manufacturer) for 18S ribosomal RNA detection
Response: Thank you so much for the suggestion. We have now added 18S rRNA primer sequence in material and method sections.
Line 138-139
Comment 2: Figure 2: The N-P panels (WT) mentioned in the text and figure legend is missing from this figure.
Response: We are so sorry for the mistakes. Figure 2 has been modified now and N-P panels is mentioned.
Comment 3: Line 271: There is no Figure 12.
Response: We are extremely sorry for the mistakes. Figure 12 is corrected and included as figure 9.
Comment 4: Line 276: Dip2b protein levels were not determined in this study (no antibodies to Dip2b are mentioned). This should be removed from the statement.
Response: Thank you for the suggestion. We have now modified the sentence. Line 291
Comment 4: Line 282-283: Please clarify this sentence about what the study found (if Dip2b is solely expressed, or if it was knocked out and function was looked at).
Response: In the present study, we found Dip2b expression in neuron while in the previous study on Dip2b knockout mice, we identified its function relation to hippocampus neuron growth. So, Dip2 was knocked out and the function was looked at. Thank you so much pointing it out. We have now added few words to clarify this sentence. Line 298
Comment 5: Line 301-302: Expression in certain tissues does not suggest a role in cell fate, only that the gene is expressed and should be removed.
Response: We have now removed the word cell fate.
Round 2
Reviewer 2 Report
The reviewer is satisfied with the modification applied. No more comments.
Author Response
Thank you so much for revising our manuscript extensively. We are glad to know that no further corrections are required.
