Wild Type C57BL/6N ES Cells
C57BL/6 is one of the best characterized inbred strains of mice and is the reference strain for the mouse genome sequence. However, genetic manipulation of the mouse genome is carried out predominatly in embryonic stem cells derived from the 129 strain of mice.
In contrast all EUCOMM ES clones generated by gene targeting have been generated in a mouse ES cell line of the genetic background C57BL/6N. Breeding of EUCOMM mutant mice with C57/6N mice allows users to perform phenotyping studies in a pure inbred background.
For the EUCOMM program a set of clonal sublines were tested grown either on feeders or on gelatin for their germline transmission potential. Two sublines in particular, JM8.F6 (feeder-dependent) and JM8.N4 (feeder-free) produced favorable results, and these lines were tested for their ability to support high-throughput gene targeting. Targeted clones derived from the JM8.F6 cell line produced a 62% germ line transmission rate while the feeder-free JM8.N4 cell line appear to be particularly proficient, producing a 69% clonal rate of germline transmission.
For the generation of the agouti ES cell lines the non-agouti locus in JM8.F6 embryonic stem cells was repaired by gene targeting. Restoring agouti function to C57BL/6N embryonic stem cells allows visualization of embryonic stem cell-derived mice by coat color and permits the recovery of pure inbred mice from test crosses with C57BL/6N mice. To assess the suitability of JM8A3 cells for high-throughput gene targeting, targeting experiments were performed and the clonal transmission rate of targeted clones were determined as discussed above. A clonal germline transmission rate of 80% was obtained from the injection of 11 targeted clones.
| Background | Substrain | Feeder | coat color | GLT rate % |
| C57BL/6N | JM8.F6 | feeder dependent | black | 62 |
| C57BL/6N | JM8.N4 | feeder independent | black | 69 |
| C57BL/6N | JM8A3 | feeder independent | agouti | 80 |
| C57BL/6N | JM8A1.N3 | feeder independent | agouti | |
| C57BL/6N | JM8A3.N1 | feeder independent | agouti |
The results of this study are presented in detail in the publication Petttit et al., 2009.
Users who would like to order one or several of the wild type ES cell sublines has to fill out the MTA of the Sanger Institute (download), have it signed and send it to Alison C.King [ ack@sanger.ac.uk ] at the Sanger Institute. As soon as the fully executed MTA has been transmitted to us, we will send out the wild type JM8 ES cells. We can be reached under the email address: info@eucomm.org
The handling fee per wild type ES cell line will be 1000€ . You will be receiving two cryovials for this handling fee: One very low passage of the cells for your electroporation experiments and one vial with cells of a higher passage for training purposes in order to get your tissue culture staff accustomed to the work with JM8 ES cells.