-->PONE-D-26-18919-->-->Germline-mediated ubiquitous recombination in ScxCre male mice: implications for tendon research-->-->PLOS One

Dear Dr. Cao,

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PLOS One

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Additional Editor Comments:

Additional comments�

1� To help the readers to understand how the complicate mouse genotyping is performed, please draw detail figures showing the position of forward and reverse primers relating to specific genetic locus or targeting constructs.

2, Please verify the accuracy of PCR primer sequences. A blast search showed a primer labeled as "RosaWT-R: 5’-CGTACTGACGGTGGGAGAAT-3" actually maps to "Cre recominase" coding sequence. Another blast search showed the primer labeled as "Ai9(floxed)-F: 5’-CTGTTCCTGTACGGCATGG-3’" matched to EGFP coding sequence. Please explain how the PCR is performed or correct it if there is a mistake.

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

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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

Reviewer #3: 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

Reviewer #3: 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: This manuscript reports an important finding for researchers conducting similar studies using ScxCre mice and related Cre-loxP systems. The observation that paternal transmission of ScxCre can lead to germline-mediated ubiquitous recombination has substantial practical implications. In this sense, the manuscript has clear value, as it may help prevent misinterpretation of experimental results and improve the efficiency and reliability of research in this field.

I also appreciate that the manuscript does not merely report an unexpected phenomenon, but places it in a broader methodological context by discussing possible causes and proposing practical breeding strategies to avoid unintended germline recombination.

However, I believe that the following points would benefit from further clarification.

First, the discussion of BAC transgenesis is important for interpreting the present findings, but this approach may not be familiar to all readers. It would therefore be helpful if the authors could provide a slightly more detailed explanation of the characteristics and limitations of BAC transgenic approaches, particularly in comparison with other genetic engineering strategies such as knock-in approaches.

Second, BAC transgenesis can occasionally result in partial integration, rearrangement, copy-number variation, or insertion-site-dependent effects, which may alter expression specificity. While the authors’ interpretation of ectopic/leaky germline Cre activity is plausible and likely correct, clarification regarding whether transgene integrity or founder-specific effects have been evaluated would further strengthen the manuscript.

Reviewer #2: In this manuscript, the authors demonstrate that ScxCre exhibits significant off-target recombination activity in the male germline, leading to ubiquitous recombination of floxed alleles in offspring independently of Cre inheritance. The study further shows that this phenomenon is specific to male transmission and is not observed upon maternal inheritance of ScxCre. Overall, the experiments are well designed and the data support the main conclusions.

This work is important for the field as it provides a critical technical caution for investigators using ScxCre lines. The finding that germline recombination can occur prior to meiosis in diploid germ cells has direct implications for breeding strategy design and interpretation of conditional knockout and lineage-tracing experiments. In particular, the study highlights the necessity of strict parental sex selection to avoid unintended global recombination events, which is highly relevant for the Cre-LoxP user community.

Minor comments:

1. In Figure 1C, three PCR products are shown for the Ai9 allele (WT, floxed, and recombined). The WT PCR shown for the Ai9 locus presumably amplifies the Rosa26 wild-type allele. If so, it is unclear why no WT band is observed in the dam and pups 1 and 2. The authors should clarify the expected genotypes and banding patterns for WT, heterozygous Ai9, and recombined alleles, as well as the primer binding strategy.

2. In the Methods, the origin of the ScxCre line is cited as Ref. 10 however, this reference does not provide a primary description of the generation of the ScxCre mouse line, only states that it will be described elsewhere. Please include the appropriate original reference describing the generation and validation of this transgenic line.

3. Please include Jackson Laboratory strain numbers for all mouse lines used in the Methods.

4. Please verify references 12 and 15, as they appear potentially duplicated.

Reviewer #3: The authors present a paper about dangers of incorrect breeding management regarding the widely used Scx-Cre mouse model, focusing on the dangers of germline recombination, when using male germline for propagation. In contrast, no spontaneous recombination was found when using female germline. This finding is an important information for adapting breeding strategies and colony management.

Although the paper is simple and technical, its findings are generally important and varrant publication.

If I were to suggest an improvement, the authors could further explore this finding in context of other, unrelated, transgene mouse strains and compare the situation, to better inform the reader of the broader context.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Germline-mediated ubiquitous recombination in ScxCre male mice: implications for tendon research

PONE-D-26-18919R1

Dear Dr. Cao,

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.

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Kind regards,

Hualin Fu

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

**********

-->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

Reviewer #3: Yes

**********

-->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

Reviewer #3: Yes

**********

-->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: (No Response)

Reviewer #2: The authors have satisfactorily addressed all of the comments raised in the previous review. The manuscript has been improved accordingly, and I have no further comments.

Reviewer #3: The authors addressed my comment appropriately as well as the other reviewers. I have no further questions and recommend the work for publication

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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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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