Transgenesis and web resources in quail

Reviewer #2:

[…] Points that would be helpful to address in more detail are.

Assuming that it may prove difficult for most researchers to set up their own quail colonies and long distance shipping may not be a realistic option, effective use of these transgenic lines will require availability through a local providers. It would be useful to outline whether this is/can be addressed.

The worldwide availability of the lines we generated and the ones to come is obviously an important issue that needs to be addressed if we want the technique to spread in the scientific community. Since transgenesis in quail is a rather recent technology, we can only talk about how a handful of colleagues that contacted us addressed this question so far. Few decided to set up small quail husbandries to maintain quail lines locally. Others decided to set up collaborations with the laboratories that generated and maintain the lines, which regularly provide fertilized eggs for their experiments. While setting up a quail husbandry locally may sound attractive, the amount of paperwork required by local and national administration, ethics and GMO committees, can be a significant deterrent as there are no established regulations on transgenesis with this novel animal model. Collaborations, on the other hand, are usually done on a small scale, and within reasonable distances from the source. To add to the confusion, international importation laws also forbid the transport of fertilized eggs between certain countries. To address those concerns, we are working on establishing reliable sources of fertilized eggs in two locations. The first is at the Veterinary school of UC Davis, CA, with whom a verbal agreement has been reached in recent months to centralize some of the existing quail lines and distribute them in the US; the second is in France, where ongoing discussions with the Lyon Veterinary School aim for something similar in Europe. Those consultations involve quite a bit of persistence and their outcomes are still uncertain. In this evolving situation, we cannot give specific details in the manuscript that address this issue, but we propose to mention those details in the QuailNet Website and refer to them in the manuscript as follows: "The worldwide availability of existing and future quail lines is currently addressed and details about this can be found in the QuailNet website".

The website at the moment primarily summarises the currently existing transgenic quail lines and some novel information on anatomy. It furthermore bundles other web resources (genomics, expression studies etc., some links not active).

In order to extend this to a real community resource it will be critical to know how it is going to be maintained and updated in the longer term.

The website has been designed in two parts: (i) a publicly accessible part (the website) and (ii) a section restricted to administrators and resource providers. Administrators will be in charge of populating content and curating submitted data. Regarding genomics, orthologues, expression data, etc., automatic scripts have been designed to ensure the regular updating of databases with the latest available information. The website is currently under a development phase and it is hoped that it will evolve as the technology spreads within the community. Major developments or changes to the existing website would however require specific fundings for a developer's salary. Upon request, the scripts/database structure can be released through a git repository so that anyone can contribute.

Can data be provided to the anatomy section/Materials and methods section? How is this going to be curated? etc.

Regarding anatomy sections, this is part of a project called http://anatomyportal.org/, led by Dr. Robert Bryson Richardson and for which Dr. David Salgado was the lead developer. Please contact Dr. Richardson (robert.bryson-richardson@monash.edu) for updates or modifications to the website.

Reviewer #3:

In general, the manuscript is well written and clearly presented with beautiful and meaningful images and videos. However, there are some aspects that should be improved:

1) The manuscript does not give any information concerning the number of the transgene copies that were inserted in each of the generated lines or how many separated F1 founders they were checking for each of the three transgenic lines described. It is not clear either, how stable will be the transgenic lines after several generations, as they only give data on the F2.

We have not tested the actual number of copies in the genome of our founders, but the transmission of the transgene to the offspring in each of the quail lines indicates that the transgenes are present in one copy. This is coherent with the observation of our collaborator (T. Doran, that has developed this technology in chicken), which showed that among the 150 founders they analysed, a large majority (64%) contained one insertion in the chicken genome, while 27% contained two insertions.

These are the number of F1 founders for each transgenic line.

TgT2(CAG:NLS-mCherry-IRES-GFP-CAAX): 3 F1 founders

TgT2(CAG:Kaede): 3 F1 founders

TgT2(Mmu.MLC1F/3F:GFP-CAAX-IRES-NLS-mCherry,Gga.CRYBB1:GFP): 3 F1 founders

This has been added to the text.

We have not observed any significant decrease of the signal over time, suggesting, as mentioned in the text that Tol2 insertions are not particularly prone to inactivation over time.

2) The manuscript discuss poorly the transposon method they have used in relation to other methods such as lentivirus or adenovirus based method in relation to the efficiency of germline transmission. For instance, adenovirus mediated CRISPR/Cas9 mediated deletion has a transmission between 2.5 to 10% in a recent article by Lee et al., 2019, or in another recent lentivirus generated line, Ubiquitous expression of membrane tethered EGFP generated by one of the labs in the manuscript, rendered a transmission of 8% (Saadaoui et al., 2020).

The comparative efficiencies of those techniques is not really a limiting factor. To take an example, if a F0 male, found positive for the transgene with our technique, is crossed with 4 females that each lay an egg-a-day, one F1 will be made every 25 days of crossing (for an efficiency of 1%). As we usually cross 3 positive F0 males, we obtain about 3 F1 in the same period. The efficiencies that have been published about viruses are definitely higher (lentivirus: 8%; adenovirus: 2.5-10% efficiency) and they could allow a shorter crossing period, but in fact, our colleagues that use viruses tend to cross males with females for just about as long a period, such that they obtain more F1 than we do, which they anyhow cull to keep only two to three. Such that practically, there is not a huge advantage at having higher efficiencies to perform transgenesis. A low efficiency may become an important bottleneck if more delicate technologies are envisaged (e.g. KI), but it is unlikely that this can be solved with viruses, since the combined sizes of the Cas9, the gRNA, their respective promoters, the homology arms, and the KI construct (e.g. GFP) are clearly out of size range for viruses. As explained in the text, there are other advantages of the plasmid technique over the virus approach that make this technique attractive for specific applications.

3) The authors should provide more details about the protocol for transgenesis: DNA quality purification, quail sperm obtaining, specific quail embryos clearing for 3DISCO.

Plasmids are prepared using the Nucleobond endotoxin-free midiprep kit. This is now indicated in the Materials and methods section.

Semen is collected from 7 week-old males using a female teaser as described in

Chelmonska et al., 2008. More details about the technique have been added to the text.

As indicated in the Materials and methods section, we followed the exact protocol of the 3DISCO procedure described in the Cell paper of Alain Chedotal's team (Belle et al., 2017) to prepare the quail embryos for IHC and 3D imaging.