FAQ About RFA-RM-10-011, RFA-RM-10-012 and RFA-RM-10-013
National Human Genome Research Institute
National Institutes of Health U.S. Department of Health and Human Services
Frequently Asked Questions about RFA-RM-10-011, RFA-RM-10-012 and RFA-RM-10-013
Knockout Mouse Phenotyping (U54)
Knockout Mouse Project Database (U54), and
Knockout Mouse Production and Cryopreservation (U42)
On Tuesday, October 12, 2010, 12 p.m. — 2 p.m. ET, an informational session was held to discuss the three KOMP2 RFAs (RFA-RM-10-011, RFA-RM-10-012, RFA-RM-10-013). There were attendees both in person and via teleconference. An overview of KOMP and KOMP2 programs, including the structure, timeline, and goals of the programs was presented by Project Officers Dr. Colin Fletcher (NHGRI), and Dr. Raymond O'Neill (NCRR). Based on questions raised during the teleconference and inquiries made to NIH staff via e-mail or direct phone calls, a compilation of frequently asked questions with answers from NIH staff is presented both as a slide set available at the session and as follows:
Did the RFAs intend the budgetary limits to be in Total Costs or Direct Costs? Is a three percent per year escalation in the out-years' budgets allowed? If subcontracts are utilized, can the dollar limits be exceeded to cover compounded F&A costs?
Total costs are the relevant figures for these RFAs, with no escalation in out-years, and the stated limits are all-inclusive.
What is the timeline for production (i.e., what is the lead time of production before beginning phenotyping)?
Currently, the KOMP repository and production Centers have been creating mice as a QC procedure for ES cell characterization. The KOMP repository now has hundreds of lines in the form of cryopreserved germplasm. The KOMP2 production centers can utilize these embryos, so we do not anticipate significant delays.
Will the IKMC Repositories be able to handle sending out an additional 500 lines per year?
The anticipated 50 or so lines per month required for KOMP2 programs are fewer lines than are currently distributed. NIH intends to work with the KOMP Repository to make sure that this transition can be made and can be made smoothly.
Who will be conducting the gene selection process and how?
The gene list will be negotiated between the PIs, the NIH program staff, and the Advisory Panel after the grants have been awarded. This will be the main topic of the kickoff meeting.
NIH staff has also heard from the mouse community that they would like uncharacterized genes selected (~20-25 percent of the total genes should be unknowns).
The gene lists will be distributed among the Production Centers.
Can we utilize ES cells that we already have in-house? What are other ES cell repositories that can be utilized for this program? Are Gene Traps and Transposons mutations acceptable as part of the program?
A KOMP2 gene prioritization committee (which will include PIs of the awards) will select which genes are produced and phenotyped first, and the type of allele will be heavily weighted in this prioritization to KOMP resources. Sources of ES KO lines other than the KOMP and EUCOMM repositories may have no conditional allele available, may have quality control problems, and may have unresolved issues with a variety of MTAs that govern use by third parties and re-deposition to the KOMP/EUCOMM embryo/germplasm repositories. If a gene targeted, conditional ready allele is available, then that ES line is preferred, as it has the potential of creating 3 alleles. Most if not all phenotyping will take place on gene deletion alleles following Cre recombination/excision to remove the heterologous promoter driving neoR expression and, in the case of CSD alleles, the critical exon. Regeneron alleles have already deleted the gene target, but will need to also undergo the Cre step to remove the heterologous promoter driving neoR expression. It is envisioned that gene traps (many of which are hypomorphs) and transposon insertions (which are essentially gene traps) will be considered when no other allele is available, but these selections must be approved by the prioritization committee that is to be set up. The ability to remove the heterologous promoter driving the selectable marker will be very important in the selection process. All ES lines should be from C57BL/6N and they must be available to the research community in keeping with KOMP guidelines and NIH Research Sharing policies.
KOMP ES cells may be usable even if they have been previously ordered for other purposes. If other ES lines are desired and they are not IKMC ES cells, additional QA/QC should be performed, subject to approval of program staff. If it turns out that the cells from an outside repository are not adequate, this information should be shared with the entire research network for discussion on whether or not these ES cells should be used in the future. All ES lines should be from C57BL/6N.
KOMP, EUCOMM, NorCOMM, TIGM, and Piggyback transposons are all available in repositories and are in C57BL/6N 6 ES cells and can be utilized for KOMP2, within the above stated guidelines and subject to prioritization by the review committee that will be formed at a later date.
KOMP calls for mutant protein encoding genes. Are micro RNA genes and other non-protein encoding genes allowed?
See Questions 4 and 5 above, about gene list prioritization and MTA issues. KOMP and IKMC alleles are expected to have priority. However, after scientific review, if your scientific explanation can support inclusion of micro RNA encoding gene targets, or other non-protein encoding gene KOs, NIH staff will carefully consider it as well.
Will production centers need to be prepared to receive clones from any/all IKMC members (more than just KOMP)?
Production centers will need to be able to accept all IKMC products, subject to discussion of MTA issues. NIH staff will address any MTA issues after award.
The RFA calls for removal of the neoR cassette for phenotyping. Do Production Centers need to preserve germline clones of multiple alleles?
NIH program staff has been debating this. Although it would be more expensive to do this additional step, it would be beneficial. It is recommended that the conditional ready allele be preserved in the germline. Whether this is done via mouse breeding or working in vitro in ES cells with multiple injections is a matter for the applicant to propose, and will therefore be addressed in the scientific review process.
Before the KOMP Repository sends out clones it performs QA/QC. Will the KOMP Repository continue to perform the QA/QC when sending clones to the centers? If so, does this mean that the centers do not have to perform their own QA/QC?
Whether the centers have to perform QA/QC is up for discussion and will be part of scientific review. In the case of KOMP repository ES lines, the repository performs specific tests that follow KOMP standards, and it would seem unnecessary for the QA/QC to be performed twice. However, it is recommended that applicants should plan to include some level of spot checking to ensure the correct identity of ES lines.
Is the ES cell QA in the KOMP Repository part of the basic clone cost?
This information is available on the KOMP Repository website.
For EUCOMM, the website suggests that a Southern blot be performed as QA/QC to confirm that the insertions are correct. What QA and QC does the production center need to perform for KOMP2?
At this time NIH staff does not believe that Southern blots should be required given the success rate in PCR screening and the cost related to Southern blots. It is strongly recommended that the ES lines should be tested at both the 5' and 3' insertion sites and that neoR counting (or Southern if preferred by the applicant) should be performed to test the integrity of the allele and test for potential additional vector insertions. The Regeneron alleles, due to the long arms of homology, are only tested for loss of allele and neoR counts. Various sources of ES lines have different levels of QA and QC testing. If the above tests are not performed by the ES repository then it is the responsibility of the Production Center to ensure that the appropriate tests are performed on all lines.
In terms of the possibility of second insertion sites, if a product has been validated by KOMP, do you need to confirm the validity of the allele?
You should get in touch with the KOMP repository regarding their success rate — you can find out how often they find random insertions. It is a very low percentage, but you can base your own grant–writing decision on these figures.
The assays that are performed at the KOMP Repository are independently designed (often distinct from the screening for targeting events), and therefore are rather objective.
Is there a standard diet that will be used at the production and phenotyping centers?
It is preferable that each of the centers agrees to use a standard diet but it is understood that institutional policy may not allow this. This is another discussion point for the research network.
In terms of breeding cohorts up to 7x7, whose budgetary responsibility is it? The production centers or the phenotyping centers?
Since you cannot be funded for more than was stated in the original grant, you should present the most expensive option and use that as a basis for your budget. You should be flexible and possibly propose different options knowing that it can be discussed later. The negotiations on the budget will occur after the grant has gone through the review process.
Who should pay for shipping of cryopreserved samples and mice from the production centers to the phenotyping centers and repository?
The center that is receiving the shipment should pay for the shipping. Production groups must produce adequate numbers of mice or embryos to provide Phenotyping Centers but the cost of shipping should be the responsibility of the Phenotyping Centers. When the Production Centers ship materials to designated repositories the cost of shipping should be borne by the repository.
Will the costs for serum testing/histology/other in vitro tests that would be done at a central facility be the responsibility of the central facility or the sender?
The repository will provide testing and health status information to the Production Centers and they in turn will provide their health status information to the Phenotyping centers. Cost of testing is the responsibility of the shipping group.
Will the phenotyping centers run liter mate or wild type controls?
C57BL/6N wild type controls are acceptable if they have been housed under the same conditions. It is anticipated that some wild type controls will be run frequently but an equal number of wild type controls for every KO line tested is not required.
How do I learn which phenotyping project focuses on liver functions/disease?
It is recommended that you check the Sanger Mouse Genetics Program, the EUMODIC, and Europhenome websites for the projects they are performing related to liver function. Applicants may put forward other and additional assays that focus on liver function and disease. The merit of all phenotyping proposals will be determined at review.
Could you please clarify what your goal is in requesting fertility and fecundity testing? It was suggested by attendees that this aspect would be extremely breeding-intensive and therefore is a phenotyping issue. It was suggested by attendees that Fertility and Fecundity should be part of the Phenotyping Centers mission and not part of the production centers.
The production centers are expected to identify homozygous lethals and track the breeding information to provide future end users with information on the breeding of KO lines.
It was envisioned that the production groups would have rapid access to homozygous mice and that this step could be included at minimal costs. The experiment requested is not meant to be a comprehensive study of fecundity but instead provide general information on fertility. This will allow, in most cases, for investigators to know which lines can or cannot be bred as homozygous lines. If a production center uses a breeding schema to utilize homozygous mice to generate cohorts, this data should be collected and presented.
Another type of study could be performed, for example, on as few as 2-3 homozygous mice per sex set up for 3-4 months. Numbers of females and males giving rise to live births, number of mice and sexes per liter, and health of the pups prior to weaning could be recorded. Alternatively, fertility could be subcontracted to a phenotyping group using breeding or other assays such as pathology.
It was previously discussed that the grants would include an aging component, where is it?
Due to monetary constraints, it was decided not to address it in these grants.
Can we propose an extended Tier 1 phenotyping like in EUMODIC?
That is acceptable and is a topic for scientific review.
Is Tier 2 phenotyping upon NIH request or will this be a center's decision?
Tier 2 phenotyping is envisioned to be another level of phenotyping and not part of the KOMP2 project as this time. Separate funding for these projects should be sought unless applicants can meet the stated goals of KOMP2, as well as Tier 2.
In terms of information resource sharing, will there be a standardized platform?
It is expected that the DCC will be hands-on in terms of developing methods to interface with each group to collect data. It is expected that each Production and Phenotyping Center will have different LIMS systems in place to extract data and deliver it to the DCC. It is also expected that the phenotyping centers will agree on data coding standards, but it will be a research network discussion and not the responsibility of the DCC alone.
What resources are supposed to be shared with the research network besides the end product?
Mice, ES cells and mouse related materials.
The progress of the project, data, metadata, information on process improvements and to work collaboratively. Please refer to the PI responsibility section of the RFA.
In terms of the DCC storing image data, is there a concept that addresses contingency?
NIH staff has done preliminary investigation to get an idea of the potential size of the data sets. NIH expects the program to preserve images and this will be a large portion of the data set. Due to the size of the data set, the DCC can propose to either have a central website that houses all the information, or a website that may point to another site that houses the data.
Once the production or phenotyping centers have handed off their data to the DCC, the centers may want to have backup data for temporary security, but overall the centers should not have to maintain the data sets permanently. The length of time to maintain backup information will be subject to discussion among the grantees and NIH staff.
What will be the moratoriums placed on the data sets of KOMP2?
All policy discussions will be addressed later on in the process but NIH staff expect rapid lease of data without a holdback period.
What is the format of choice for uploading the data from the phenotyping center to the database?
To be determined post-award but the DCC RFA provides some general guidance.
Will the DCC be responsible for tracking data from the Production Centers as well as the Phenotyping Data?
Yes, the RFA states:
6.I.1. Data Coordination Center Database: The data being tracked by the DCCDB should include, but are not limited to, a variety of information types relating to the production status of mutants (from ES cell selection through to production of mice) and the locations of ES cells or mice in NIH-supported repositories. It will compile and track several lists of mouse genes — those that have been mutated and for which ES cells, frozen embryos, sperm or mice are available to investigators, those that have entered into the mutation production and phenotyping pipelines and their status in the pipeline, and those that remain to be taken up by the pipeline. This functionality must allow scanning across the database not just the following of individual mice. Metadata collection, including a timestamp on all data sets will be vital to allow proper tracking of progress and retrospective analysis of the projects".