Last updated: June 28, 2011
Development and Improvement of Inbred ES Cell Lines for Use in Generation of Mouse Mutants DA-06-009
Frequently Asked Questions (FAQs)
- What characteristics define robust C57BL/6 ES cell lines according to the goals of RFA-DA-06-009?
The goal of the RFA is to create C57BL/6 ES cell lines whose cost for high throughput gene targeting or insertional mutagenesis is cost effective within the guidelines of KOMP's goal to complete the production of a null mutation for each gene in the mouse genome (RFA-HG-05-007, "Completion of a Comprehensive Mouse Knockout Resource"). It is anticipated that the cost for C57BL/6 ES production cell lines may be higher than for 129 derived ES cells, but these cost should not preclude the completion of the KOMP goal. The main criteria for comparing cost and efficiencies between C57BL/ES cells are the:
A) Number of colonies and frequency of targeting events post transfection
B) Ease/efficiency (time and success rate) of colony picking (post selection), replating and freezing
C) Efficiency to thaw, expand and to make chimeras
D) Frequency of germline transmission
E) Ability to maintain totipotency and high germline efficiency through multiple passages
F) Ability to produce enough vials of C57BL/6 ES cells to complete the KOMP project
- Is the use of BAC derived targeting vectors required by the RFA?
Although the RFA states that a desired characteristic is the ability to do gene targeting using BAC vectors, any methods may be used to validate the robustness of the C57BL/6 ES cell line. Thus, any approaches for producing targeted or insertional null mutations will be considered responsive to the RFA.
- Is it an absolute requirement of the RFA that the C57BL/6 ES cell line be feeder cell independent?
No. If you can demonstrate that the cost and efficiency to produce C57BL/6 derived ES Cells for high throughput gene targeting or insertional mutagenesis on feeder lines is cost effective within the scope of the KOMP goal to complete a null resource for the mouse genome, then the proposal will be considered responsive to the RFA.
- May we propose to use a C57BL/ES cell line that we have already created for the purpose of this RFA DA-06-009?
One of the goals of this RFA is to obtain validated and robust C57BL/ES cells for the entire Knockout Mouse Project, particularly for use in the RFA HG-05-007, "Completion of a Comprehensive Mouse Knockout Resource." Applications proposing C57BL/6 ES cells that have already been validated in terms of the goals of RFA DA-06-009 will not be considered responsive. If you have such a line please contact us directly. Applications using existing C57BL/6 ES cell line will only be considered responsive, if specific aims of the proposal are to define, expand and improve culture conditions for growing the existing C57BL/6 ES cell lines, improve the efficiency and decrease the cost of using the existing C57BL/6 ES cell line, and/or validate the rate of germ line transmission. Production of additional lines as back-ups or to attempt further improvements can be included in the application.
- What are the expectations about the sharing and distribution of the ES cells and the 50 genetically modified germline strains needed to validate the C57BL/6 ES cell line?
The successful applicant will be required to transfer all data and resources pertaining to ES cells and mice generated by this initiative to the centers involved with the efforts funded under RFA-HG-05-007, "Completion of a Comprehensive Mouse Knockout Resource", to an NIH-funded central repository or an NIH-funded central data base, whichever applies. Therefore, responses to this RFA should propose a specific and comprehensive plan for the rapid release of data and materials resulting from research to develop improved C57BL/6 ES cell lines to the appropriate databases and repositories, respectively. The quality of this plan will be an important criterion in the review of the application, and an appropriate plan for release of data and materials will be made a condition of the awards made as a result of this RFA.
In presenting the data release plan, the release of data to the project's public tracking website must be discussed and should include information for any ES cells or mice that contain gene targeted mutations made during the developmental effort including, but not limited to, information identifying the genes that have been mutated, verification that the construct sequence has been sent to dbGSS, data that confirm a null mutant has been successfully made, information that the ES cell has been deposited in a public resource designated by NIH and its repository location, and finally that a mouse has been made from the mutant ES cell line and its repository location (for the small number that will be made under this RFA as a quality control measure). The materials release plan should account for the release of materials generated as part of the developmental activities and include the release of targeting vectors, mutant ES cells, and any knockout mice, including frozen embryos and sperm samples, derived in the course of the supported work
- Do the 50 KO lines all have to be genes not previously inactivated?
It is preferred but high priority genes that are in high demand for an inbred C57BL/6 background can be included in consultation with the KOMP prioritization committee.