PONE-D-24-27880Expression of transgenic biotin ligases in inducible neuronal murine cell lines by integration into the mHipp11 gene locusPLOS ONE

Dear Dr. Jansen,

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Academic Editor

PLOS ONE

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RPJ was funded by a grant of the Deutsche Forschungsgemeinschaft (grant no. DFG JA696/10-2) in the course of the DFG-funded Research Unit FOR2333 (https://gepris.dfg.de/gepris/projekt/270067186).

LF was supported by the International Max Planck Research School “From Molecules to Organisms” (https://www.phd.tuebingen.mpg.de/imprs).

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  Originality and Relevance: The study addresses a significant problem in the field of genetic engineering and cellular biology by exploring a method to ensure consistent expression of transgenes during cell differentiation. This is relevant for researchers working on gene expression in stem cells and differentiation studies.

Technical Quality and Rigorous Methodology: The methodology is robust, employing multiple experimental approaches to validate findings. The use of RT-qPCR, Western blotting, and immunofluorescence, biotinylation assay provide a comprehensive validation of results.

Interpretation and Validity: The interpretation of results is logical, and the conclusions are supported by the data presented. The study provides clear evidence that targeting the mHipp11 locus can minimize transgene silencing after differentiation.

Based on the evaluation, I recommend accepting the publication with minor revisions. The study is well-conducted and addresses an important issue in the field, providing valuable insights and a potential solution for transgene silencing during cell differentiation. However, the following minor revisions are suggested to enhance the clarity of the manuscript.

Please identify the clones used for subsequent experiments and please label them accordingly in both the figures, figure legends and the text. As of current, I can only see Clone 11 and Clone 15 data mentioned in Figure 2B. I request the authors to identify to the clones used in the subsequent Figures 2C, 3, 4.

Please also comment on the transgene silencing effects comparing Clone 11 and Clone 15, ideally providing data, if available.

Concern: The DAPI channel exposure for figure 2C (3h) is noticeably higher than the rest.

Reviewer #2:  In this manuscript, Feicht et al. showed that the Hipp11 locus can be utilized in combination with other transgenes located in other safe harbors. The authors present results showing that the Hipp11 safe harbor is a more reliable and less silenced locus than in the random integrations strategy. Although most of the data appears convincing, there are still a few major and minor comments that have to be addressed by the authors to strengthen the overall manuscript.

Major comments:

- The authors presented qPCR data of the MicroID expression in Figure 4B. However, the total protein level of MicroID fusion is still missing for mESC, Neuron Day 8, and Neuron Day 10 cells. Also, did the authors test the levels of MicroID longer than ten days to confirm (or not) that the silencing is not happening later?

- Similarly, it will be critical to evaluate the levels and subcellular localization of MicroID in mESC and neurons. This will help confirm the biotin labeling as well. Showing GFP images in live cells might show unwanted autofluorescence and background. Immunostaining the GFP fusion can help enhance the specific signal and lessen the non-specific background, especially in a non-GFP channel.

- The authors will have to validate the pluripotency markers of the clone(s) used in Figure 3. After differentiation, neuronal markers have to be used to validate the derived neurons.

- In Figure 3C, right panel, the images in red are not convincing. They will need to be shown with higher resolution, ideally co-stained with the MicroID fusion protein.

- In Figure 3A, the authors stated that the wild-type (WT) cells show no nuclear staining. Fig 3A lacks DNA staining to be able to conclude with confidence the exact subcellular localization. Also, WT cells already display GFP-positive accumulation likely in the cell body. These issues will have to be addressed.

- Conclusions in lines 334–342: The authors will have to re-formulate these conclusions since we still see a very faint biotinylation effect coming from potential active biotin ligase in + biotin conditions in random genomic integration for mPGK MicroID and CMV TurboID (Fig4C).

Minor Comments:

- We do not see in the result section which clone(s) the authors followed up with in Figure 3.

- The authors will have to perform statistical analysis for fig4B and quantify the levels of AF594 for fig3C (or the updated fig3C).

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Reviewer #1: Yes:  Arthur Luhur

Reviewer #2: No

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PONE-D-24-27880R1Expression of transgenic biotin ligases in inducible neuronal murine cell lines by integration into the mHipp11 gene locusPLOS ONE

Dear Dr. Ralf-Peter Jansen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised by Reviewer #2.

Please submit your revised manuscript by Dec 28 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Chunming Liu

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All comments have been addressed. Both figure and text revisions have been completed as requested and this has improved the clarity and quality of report.

Reviewer #2: The authors addressed most of the comments. Below are a few minor follow-ups to finalize this review.

Minor Comments:

- The S5 supplementary figure shows red saturated bands. Unless this is required by the journal, these blots are better shown in grayscale.

- The S6 fig is not the best quality wise; it would be still interesting and important to show this data as a supplementary figure and briefly touch on this challenging aspect in the text, so the scientific community is aware of such information.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Expression of transgenic biotin ligases in inducible neuronal murine cell lines by integration into the mHipp11 gene locus

PONE-D-24-27880R2

Dear Dr. Ralf-Peter Jansen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Chunming Liu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: No

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