Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2–Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2–Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

brands/companies. 3. Line 115: "all kinds of tissues" is overstated. The authors only investigated liver and muscle from transgenic and wild-type mice. 4. Line 145, Line 146, Line 176: "various tissues" and "transgenic tissues" are not precise. Figure  1c and 2c only show the RT-PCR results of liver. 5. Line 152, Line 394, Line 395 and Figure 1d-1e: The authors talked about PUFA analysis of liver samples in F0 transgenic mice in the text, but the legend of Figure 1d showed it was muscle samples, and it was unclear of which samples used in Figure 1e. Same mistakes were found in Figure 2d-2e. The authors need to display the PUFA analysis results of both samples (i.e. liver and muscle) in Figure 1 and 2, and mark the sample name in the figures. 6. Figure 1e, 2e, 3d and 3e: The results of PUFA analysis in Figure 3d and 3e have error bars, but not shown in Figure 1e and 2e. The authors should not omit this. 7. What is the difference in the results of PUFA analysis between F0 and F1 transgenic mice? The authors need to address this critical point. 8. There are a few grammar and word mistakes that should be corrected. We recommend that the manuscript is edited by a native speaker, to improve its flow and clarity.

05-Aug-2019
Dear Professor Li, We are writing to inform you that the Editor has reached a decision on your manuscript RSOB-19-0140 entitled "Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids", submitted to Open Biology.
As you will see from the reviewer's comments below, there are a number of criticisms that prevent us from accepting your manuscript at this stage. The reviewer suggests, however, that a revised version could be acceptable, if you are able to address their concerns. If you think that you can deal satisfactorily with the suggestions, we would be pleased to consider a revised manuscript.
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Sincerely,
The Open Biology Team Reviewer Comments to Author(s): Referee: Comments to the Author(s) Comments: "Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids" by Song et al. generated transgenic mice that expressed plant gene Fad2 and Fad3, and thus could produce n-6 and n-3 PUFAs. There are my major concerns mentioned below.
1. Line 94: Readers need to know the plant samples you used to amplified Fad2 and Fad3, and the method to extarct the plant RNA. The authors should add these details. 2. Line 97, Line 109, Line 110, Line 116: Thermo, Promega, TAKARA are not Chinese brands/companies. 3. Line 115: "all kinds of tissues" is overstated. The authors only investigated liver and muscle from transgenic and wild-type mice. 4. Line 145, Line 146, Line 176: "various tissues" and "transgenic tissues" are not precise. Figure  1c and 2c only show the RT-PCR results of liver. 5. Line 152, Line 394, Line 395 and Figure 1d-1e: The authors talked about PUFA analysis of liver samples in F0 transgenic mice in the text, but the legend of Figure 1d showed it was muscle samples, and it was unclear of which samples used in Figure 1e. Same mistakes were found in Figure 2d-2e. The authors need to display the PUFA analysis results of both samples (i.e. liver and muscle) in Figure 1 and 2, and mark the sample name in the figures. 6. Figure 1e, 2e, 3d and 3e: The results of PUFA analysis in Figure 3d and 3e have error bars, but not shown in Figure 1e and 2e. The authors should not omit this. 7. What is the difference in the results of PUFA analysis between F0 and F1 transgenic mice? The authors need to address this critical point. 8. There are a few grammar and word mistakes that should be corrected. We recommend that the manuscript is edited by a native speaker, to improve its flow and clarity.

Comments to the Author
The authors have addressed the concerns raised by my review. So it can be accepted for publication.

20-Sep-2019
Dear Professor Li We are pleased to inform you that your manuscript entitled "Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids" has been accepted by the Editor for publication in Open Biology.
If applicable, please find the referee comments below. No further changes are recommended.
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Thank you for your fine contribution. On behalf of the Editors of Open Biology, we look forward to your continued contributions to the journal. Dear Editor, I, along with my coauthors, would like to re-submit the attached manuscript entitled "Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids" as a Research Article in the Open Biology. The manuscript ID is RSOB-19-0140.
The manuscript has been carefully rechecked and appropriate changes have been made in accordance with the reviewers' suggestions. The responses to their comments have been prepared and attached herewith. The revised manuscript has been checked by a native English speaker to ensure the language quality of our manuscript. Furthermore, the paper has been edited by Charlesworth Language Editing Services, and the CERTIFICATE is shown below.
We thank you and the reviewers for your thoughtful suggestions and insights, which have enriched the manuscript and produced a more balanced and better account of the research. We hope that the revised manuscript is now suitable for publication in Open Biology.
Thank you for your consideration. I look forward to hearing from you. Sincerely,

Manuscript ID: RSOB-19-0140
First of all, we thank both reviewers for taking the time to read and comment on our manuscript. We also thank the Editor for their clear guidance.

Response to Reviewer:
Comments: "Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids" by Song et al. generated transgenic mice that expressed plant gene Fad2 and Fad3, and thus could produce n-6 and n-3 PUFAs. There are my major concerns mentioned below.

Response:
We greatly appreciate the reviewer's valuable comments regarding our manuscript.
The manuscript has been completely rewritten and further edited by an English native speaker as recommended.

Comment 1:
Line 94: Readers need to know the plant samples you used to amplified Fad2 and Fad3, and the method to extarct the plant RNA. The authors should add these details.

Response 1:
We apologize for this mistake and thank the reviewer for bringing it to our attention.
We have added a description of the Fad2 and Fad3 vector in the revised "Materials and Methods" section.

Response 2:
We apologise for this careless mistake. In the revised manuscript, companies mentioned in the "Materials and Methods" include the name of the company, and the relevant city, state/province, and country.

Comment 3:
Line 115: "all kinds of tissues" is overstated. The authors only investigated liver and muscle from transgenic and wild-type mice.

Response 3:
We agree that the phrase "all kinds of tissues" overstated the original number of investigated tissue types. We apologize for the results improper description.
The newly revised sentences are displayed in Response 5.

Response 4:
Thanks to carefully read our manuscript. We agree that these sentences were not precise. In accordance with your suggestion, we re-performed the RT-PCR assay in seven major tissues of the In the revised manuscript, the new sentence reads as follows: (revised manuscript page 8-9, line 174-177) "To assess the potential expression of the Fad3 gene in vivo, we extracted the total RNA from F0 and F1 transgenic mice, and analyzed the mRNA by reverse transcription PCR (RT-PCR). As expected, the Fad3 mRNA could be detected in the skeletal muscle, fat, heart, liver, spleen, lung, and kidney of the F0 and F1 transgenic mice (response Figure 1A Figure 1d showed it was muscle samples, and it was unclear of which samples used in Figure   1e. Same mistakes were found in Figure 2d-2e. The authors need to display the PUFA analysis results of both samples (i.e. liver and muscle) in Figure 1 and 2, and mark the sample name in the figures.
We appreciate your insightful comment.
For the statistical results, we re-calculated all the experimental data, and modified the figure legends to improve clarity by adding information regarding the experimental approach.

Comment 6:
Figure 1e, 2e, 3d and 3e: The results of PUFA analysis in Figure 3d and 3e have error bars, but not shown in Figure 1e and 2e. The authors should not omit this.

Response 6:
We thank the reviewer for bringing this to our attention. We re-analysed the data of Figure 1~4, and relabeled the significance superscript. Moreover, we added the source data in the "Supplementary Material" section (response Figure 2B; 2E-F; revised manuscript electronic supplementary material, table S3-4).

Comment 7:
What is the difference in the results of PUFA analysis between F0 and F1 transgenic mice? The authors need to address this critical point.

Response 7:
Thank you for raising this point. In accordance with your suggestion, we have added new PUFA analysis experiments to the revised manuscript.
➢ For the Fad3 single transgenic mice: (revised manuscript page 9, line 190-196) "Moreover, the ratio of n-6/n-3 PUFAs in the F1 transgenic mice was similar to the ratio in F0 (skeletal muscle: from 2.62 to 0.46, P < 0.05; fat: from 2.41 to 0.57, P < 0.05; heart: from 2.77 to 0.61, P < 0.05; liver: from 1.93 to 0.55, P < 0.05; spleen: from 2.76 to 0.98, P < 0.05; lung: from 2.31 to 0.62, P < 0.05; and kidney: from 2.13 to 0.47, P < 0.05; response Figure 2C; revised manuscript figure 2c). These results indicate that the plant Fad3 gene can be functionally expressed in the major tissues of F0 and F1 transgenic mice and Fad3 play an active role in conversion of n-6 into n-3 PUFAs." ➢ For the Fad2-Fad3 double transgenic mice: 1.(revised manuscript page 11, line 234-239) "To assess the function of the Fad2-Fad3 enzymes in the F0 and F1 transgenic mouse, the PUFA content in the major tissues were compared, including skeletal muscle, heart, liver, spleen, lung, kidney, and fat. response Figure 2E; revised manuscript figure 4b displays the PUFAs profile of seven tissues from F0 and F1 of the Fad2-Fad3 mouse. It shows that all the n-6 and n-3 PUFAs were higher in compared with wild-type mice, indicating that the Fad2-Fad3 double transgenic mice had efficiently converted the monounsaturated fatty acids into n-6 and n-3 PUFAs in their bodies." 2. (revised manuscript page 12, line 253-258) "The seven major tissues from Fad2-Fad3 double transgenic mice were collected and analyzed for n-6/n-3 PUFAs ratio. Notably, F0 and F1 double transgenic mice showed a substantially lower n-6/n-3 ratio in all tissues examined compared with wild-type mice ( Figure 3A, below; revised manuscript figure 5b)."