Mammary Intraductal Gene Electroporation (MIGE): A Novel Non-Viral Gene Delivery Method Targeting Murine Mammary Epithelial Cells
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript entitled “Mammary Intraductal Gene Electroporation (MIGE): A Novel Non-Viral Gene Delivery Method Targeting Murine Mammary Epithelial Cells” introduces a promising non-viral strategy for targeted gene delivery to murine mammary ductal epithelial cells using intraductal plasmid injection followed by electroporation. The study is clearly written, and the methodology is generally well described. Addressing the points below would further improve the rigor and clarity of the work.
- The authors describe using gentle oral pressure to deliver plasmid DNA through a glass capillary. Have the authors considered employing a microinjection system to enhance the reproducibility and precision of the injected volume?
- For MIGE-treated samples, only GFP-positive regions were selected for immunohistochemical analysis. The manuscript should clarify how corresponding areas were selected in control tissues to ensure fair comparison.
- It would strengthen the manuscript to include a quantitative comparison, such as a bar plot showing the percentage of GFP-positive areas, across samples treated with the two electroporation methods and the control group.
- While GFP expression appears localized to ductal epithelial cells, validation using an epithelial-specific marker would help confirm the targeted cell population.
- The GFP signal intensity varies across individual cells in Figure 3. The authors should discuss possible sources of this variability.
- Please clarify whether the pCE-29 vector used in this study supports transient or stable in vivo expression of EGFP.
- The manuscript would benefit from a description or quantification of off-target EGFP expression observed in adipocytes of MIGE-treated animals.
Author Response
We sincerely appreciate your detailed and constructive comments. Your suggestions, particularly regarding the quantification of transfection efficiency and the validation of target cells using specific markers, have significantly strengthened the rigor of our study. We have addressed your points as follows:
- The authors describe using gentle oral pressure to deliver plasmid DNA through a glass capillary. Have the authors considered employing a microinjection system to enhance the reproducibility and precision of the injected volume?
Response: As suggested, the use of a micromanipulator is one of the options for intratubal injection. However, this apparatus is expensive, and in some sense, it's inconvenient. Instead using this system, we chose a more convenient method, based on manually injection approach using a breath-controlled glass micropipette. This allows to ensure safety (tactile feedback prevents ductal rupture) and precision. In the revised text, the following phrase is shown to ensure exact dosing by pre-loading a calibrated 10 µL droplet into the capillary and injecting the entire volume (please see lines 109-111 and 301-308 in the revised text).
- For MIGE-treated samples, only GFP-positive regions were selected for immunohistochemical analysis. The manuscript should clarify how corresponding areas were selected in control tissues to ensure fair comparison.
Response: For checking the fluorescence in the control tissues (wild-type; uninjected tissues), we harvested the region that anatomically corresponds to the site (as an experimental site) electroporated to ensure fair comparison. These points are mentioned in the revised text (please see lines 165-168).
- It would strengthen the manuscript to include a quantitative comparison, such as a bar plot showing the percentage of GFP-positive areas, across samples treated with the two electroporation methods and the control group.
Response: This is an important aspect for our paper. Based on this suggestion, we performed a quantitative analysis toward transfection efficiency. As shown in the new Figure 3C, the MIGE method achieved approximately 0.75% efficiency, whereas the WT control showed no signal (with 0% efficiency) (please see lines195-202 and 253-256 in the revised text).
- While GFP expression appears localized to ductal epithelial cells, validation using an epithelial-specific marker would help confirm the targeted cell population.
Response: As pointed out by the reviewer, we performed double immunofluorescence staining for EGFP and cytokeratin 8 (CK8) (which is one of the epithelial cell-specific markers). The new Figure 3B demonstrated that EGFP colocalizes with CK8. This finding indicates the success of targeted delivery to epithelial cells lining a mammalian milk duct in our system (please see lines 184-193 and 240-244 in the revised text).
- The GFP signal intensity varies across individual cells in Figure 3. The authors should discuss possible sources of this variability.
Response: The variation of EGFP signal intensity in the gene-introduced ducts may be attributed to the difference in the copy number of plasmids introduced or that in the cell cycle stage of each cell. These points are mentioned in the revised text (please see lines 333-335).
- Please clarify whether the pCE-29 vector used in this study supports transient or stable in vivo expression of EGFP.
Response: The pCE-29 vector used here is a typical mammalian expression vector employing a CAG promoter. It lacks no elements required for genomic integration of a plasmid. These points are mentioned in the revised text (please see lines 90-92).
- The manuscript would benefit from a description or quantification of off-target EGFP expression observed in adipocytes of MIGE-treated animals.
Response: As pointed out, we observed off-target expression in adipocytes after MIGE. Its distribution was sporadic and spatially restricted to the immediate vicinity of the ducts, likely due to minor leakage. These points are mentioned in the revised text (please see lines 245-253 and 335-338).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study presents an interesting and technically novel approach for non-gene delivery using Mammary Intraductal Gene Electroporation. However, the manuscript lacks important details in several areas.
1) Appropriate controls are missing - lack of positive and negative controls for comparison.
2) What is the transfection efficacy? Please provide quantitative analysis of CAG promoter using flow cytometry or other techniques.
3) Highlight the potential risk factors and challenges associated with this method.
4) Please provide safety, tolerability, and toxicity studies to support claim.
5) How were the electroporation parameters decided for the study? Please provide fluorescence microscopy imaging parameters.
6) How would the MIGE approach be used towards lactating breasts? Or for breast cancer treatments.
7) Minor comment: Figure 2 panel 'surgical approach' should say 'high magnification.
Author Response
We would like to thank you for your valuable feedback. Your questions regarding the safety data, control groups, and future perspectives were very insightful and helped us to clarify the experimental design and broaden the scope of our discussion. Our responses are listed below:
- Appropriate controls are missing - lack of positive and negative controls for comparison.
Response: In this study, we used untreated wild-type mice which were served as uninjected negative controls. The tissue harvested from the uninjected mice corresponds to the same site used for electroporation-based gene delivery (please see lines 165-168 in the revised text).
- What is the transfection efficacy? Please provide quantitative analysis of CAG promoter using flow cytometry or other techniques.
Response: We used IHC-based cell counting instead of using flowcytometry, because in the former case, spatial context is well preserved and cell loss during dissociation can be avoided. After evaluation of the immune-stained epithelial cells, we found that those cells were transfected with 0.75% efficiency, which is shown in Figure 3C in the revised text (please see lines 253-256).
- Highlight the potential risk factors and challenges associated with this method.
Response: In the Discussion section, we described potential risks associated with MIGE, including ductal rupture due to the accidental deep puncture and minor risks associated with anesthesia and surgery. These points are mentioned in the revised text (please see lines 355-358).
- Please provide safety, tolerability, and toxicity studies to support claim.
Response: The survival rate after MIGE was 100%. We added Supplemental Figure S1 (H&E staining), in which tissue architecture is well preserved with no overt severe inflammation. These points are mentioned in the revised text (please see lines 210-215).
- How were the electroporation parameters decided for the study? Please provide fluorescence microscopy imaging parameters.
Response: Electroporation (EP) parameters were optimized by checking the EGFP-derived fluorescence and the presence/absence of tissue damage under a fluorescent microscope. As a result, we found that low voltage (30 V) was suitable for transfection of the mammalian tissues exposed. In the case of gene delivery toward the skin under which mammalian ducts are included, relatively higher voltage (70 V) was required. When the former EP condition was applied to the exposed tissues, consistent fluorescence imaging settings were ensured for all samples. These points are mentioned in the revised text (please see lines 158-159 and 324-327).
- How would the MIGE approach be used towards lactating breasts? Or for breast cancer treatments.
Response: These questions sound reasonable. Applications of MIGE to lactating glands for secreting milk protein and breast cancer research for targeted tumorigenesis or its suppression are the next stage theme. These points are mentioned in the revised text (please see lines 367-373).
- Minor comment: Figure 2 panel 'surgical approach' should say 'high magnification.
Response: Thank you. We have corrected the label to “high magnification” in the Figure 2 of the revised text.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for incorporating all the recommended changes. The revisions adequately address my comments, and the manuscript looks good in its current form.
