Reviewer ’s report  
 
Title: Development of a transgenic mouse model of hepatocellular carcinoma with a liver  
fibrosis  background  
 
Version: 0 Date: 04 Aug 2015  
 
Reviewer: Bruno Christ  
 
Reviewer's report:  
The manuscript by Chung et al describes a novel HCC model in the mouse using stable 
transfection of c -myc and shRNA to silence p53 after hydrodynamic injection. They used this 
model to show that tumor formation was increased and survival decreased in the background of 
fibrosis induced by CCl4 treatment. Indeed, it is a short and easy way to generate mice with liver 
tumors. But at this  point, the study suffers from a preliminary characterization of the tumors as 
compared to other tumor models like DEN -induced liver tumors and confirmation in another 
fibrosis model avoiding toxic compounds.  
The authors show GFP expression in tumors and c onclude that therefore the tumors derived from 
transgenic hepatocytes overexpressing myc and silenced p53. It would be nice to show whether 
this is really the case by showing RNA or protein data. Otherwise, integration of the plasmid in a 
random genomic pl ace could also be responsible for tumor induction.  
The experiment described in Fig. 2 induces cirrhosis in the background of existing tumors. "In 
real life", it is the other way round. What happens after inducing fibrosis first by CCl4 and then 
injecting t he tumorigenic plasmids?  
It seems that in this experiment another control should have been run. Control animals should 
have been injected with the plasmid mycGFP instead of mycP53GFP and then treated with CCl4 
or not.  
The quality of the pictures is a bit poor, which might result from the conversion into a pdf -file. 
Nonetheless, differences in GFP and alpha -SMA stains are hardly visible if at all in Fig. 4.  
The authors conclude that fibrosis induced by CCl4 treatment decreased survival. Yet, it is not 
clear  whether the decrease in the survival rate is not due to a toxic impact of CCl4 even after 
long-term treatment. Therefore, similar experiments with another fibrosis model like bile duct 
ligation or DEN would be necessary to support this conclusion.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional  statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or s hares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
I declare that I have no compe ting interests  
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be poste d on the website along with the authors' 
responses. I agree for my report to be made available under an Open Ac cess Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be inclu ded in my named report can be included as confidential comments 
to the editors, which will not be published.  
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Development of a transgenic mouse model of hepatocellular carcinoma with a liver  
fibrosis  background  
 
Version: 0 Date: 04 Aug 2015  
 
Reviewer: Bruno Christ  
 
Reviewer's report:  
The manuscript by Chung et al describes a novel HCC model in the mouse using stable 
transfection of c -myc and shRNA to silence p53 after hydrodynamic injection. They used this 
model to show that tumor formation was increased and survival decreased in the background of 
fibrosis induced by CCl4 treatment. Indeed, it is a short and easy way to generate mice with liver 
tumors. But at this  point, the study suffers from a preliminary characterization of the tumors as 
compared to other tumor models like DEN -induced liver tumors and confirmation in another 
fibrosis model avoiding toxic compounds.  
The authors show GFP expression in tumors and c onclude that therefore the tumors derived from 
transgenic hepatocytes overexpressing myc and silenced p53. It would be nice to show whether 
this is really the case by showing RNA or protein data. Otherwise, integration of the plasmid in a 
random genomic pl ace could also be responsible for tumor induction.  
The experiment described in Fig. 2 induces cirrhosis in the background of existing tumors. "In 
real life", it is the other way round. What happens after inducing fibrosis first by CCl4 and then 
injecting t he tumorigenic plasmids?  
It seems that in this experiment another control should have been run. Control animals should 
have been injected with the plasmid mycGFP instead of mycP53GFP and then treated with CCl4 
or not.  
The quality of the pictures is a bit poor, which might result from the conversion into a pdf -file. 
Nonetheless, differences in GFP and alpha -SMA stains are hardly visible if at all in Fig. 4.  
The authors conclude that fibrosis induced by CCl4 treatment decreased survival. Yet, it is not 
clear  whether the decrease in the survival rate is not due to a toxic impact of CCl4 even after 
long-term treatment. Therefore, similar experiments with another fibrosis model like bile duct 
ligation or DEN would be necessary to support this conclusion.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional  statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or s hares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
I declare that I have no compe ting interests  
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be poste d on the website along with the authors' 
responses. I agree for my report to be made available under an Open Ac cess Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be inclu ded in my named report can be included as confidential comments 
to the editors, which will not be published.  
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Development of a transgenic mouse model of hepatocellular carcinoma with a liver  
fibrosis  background  
 
Version: 0 Date: 04 Aug 2015  
 
Reviewer: Bruno Christ  
 
Reviewer's report:  
The manuscript by Chung et al describes a novel HCC model in the mouse using stable 
transfection of c -myc and shRNA to silence p53 after hydrodynamic injection. They used this 
model to show that tumor formation was increased and survival decreased in the background of 
fibrosis induced by CCl4 treatment. Indeed, it is a short and easy way to generate mice with liver 
tumors. But at this  point, the study suffers from a preliminary characterization of the tumors as 
compared to other tumor models like DEN -induced liver tumors and confirmation in another 
fibrosis model avoiding toxic compounds.  
The authors show GFP expression in tumors and c onclude that therefore the tumors derived from 
transgenic hepatocytes overexpressing myc and silenced p53. It would be nice to show whether 
this is really the case by showing RNA or protein data. Otherwise, integration of the plasmid in a 
random genomic pl ace could also be responsible for tumor induction.  
The experiment described in Fig. 2 induces cirrhosis in the background of existing tumors. "In 
real life", it is the other way round. What happens after inducing fibrosis first by CCl4 and then 
injecting t he tumorigenic plasmids?  
It seems that in this experiment another control should have been run. Control animals should 
have been injected with the plasmid mycGFP instead of mycP53GFP and then treated with CCl4 
or not.  
The quality of the pictures is a bit poor, which might result from the conversion into a pdf -file. 
Nonetheless, differences in GFP and alpha -SMA stains are hardly visible if at all in Fig. 4.  
The authors conclude that fibrosis induced by CCl4 treatment decreased survival. Yet, it is not 
clear  whether the decrease in the survival rate is not due to a toxic impact of CCl4 even after 
long-term treatment. Therefore, similar experiments with another fibrosis model like bile duct 
ligation or DEN would be necessary to support this conclusion.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional  statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or s hares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
I declare that I have no compe ting interests  
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be poste d on the website along with the authors' 
responses. I agree for my report to be made available under an Open Ac cess Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be inclu ded in my named report can be included as confidential comments 
to the editors, which will not be published.  
I agree to the open peer review policy of the journal  
assistant