Hinxton, UK – Researchers from the Wellcome Trust Sanger Institute, the University of California at Davis, and Harvard University have isolated stable, highly germline-competent embryonic stem (ES) cells from the C57BL/6N genetic background. These cells are ideal for high-throughput genetic manipulation and are the foundation for two large-scale knockout programmes that will provide targeted C57BL/6 ES cells for the scientific community.
The utility of these cells was further improved by re-activating the expression of a dominant coat colour gene (agouti). This genetic change permits the visual assessment of ES cell contribution to the germline and simplifies the breeding required to generate pure inbred lines of mice.
Historically genetic manipulation of the mouse genome has been carried out mainly in embryonic stem cells derived from the 129 strain of mice. While 129 ES cells have proven to be very robust, many genetic experiments are better carried out in C57BL/6 mice, one of the best characterised inbred strains of mice and the reference strain for the mouse genome sequence.
With the completion of the mouse genome, international programmes have started that aim to knockout more than 21,000 mouse genes in C57BL/6 inbred mice. To make this feasible, robust, highly germline competent ES cells derived from C57BL/6 are required to support large-scale mouse production and phenotyping efforts that are to follow.
In their quest for suitable C57BL/6 embryonic stem cells, the team established a male cell line (JM8) from the C57BL/6N sub-strain that contained a normal complement of chromosomes and exhibited normal undifferentiated morphology when cultured on feeder cells and on gelatine-treated plates. When early passage JM8 cells were injected into blastocysts from albino mice, pups were produced that showed high coat colour chimaerism, a sex distortion in favour of males (80%) and a high proportion of chimaeras with 100% contribution to both somatic tissues and the germline.
Two further selected sub-lines, JM8.F6 (feeder-dependent) and JM8.N4 (feeder-free) were then tested for their performance in high-throughput gene targeting experiments. In these experiments the clonal germline transmission rate was reliably better than 65%. Critically, culturing JM8 cells under feeder-free conditions did not compromise their pluripotency.
Experience has shown that injection of C57BL/6N embryonic stem cells into albino (C57BL/6 Tyrc-Brd) blastocysts is a particularly favourable combination for germline transmission. A disadvantage of this combination, however, is that mice of a mixed genetic background are produced in testcrosses to albino mice (C57BL/6 Tyrc-Brd X C57BL/6N [F1]). Thus, to simply the breeding strategy required to obtain pure C57BL/6N inbred mice, the non-agouti mutation was repaired in JM8 cells.
The non-agouti mutation in C57BL/6 strains is due to an 11.8 Kb pair retrotransposon in the first intron of the agouti gene which abolishes expression. The team realised that restoring agouti function to C57BL/6N embryonic stem cells would allow visualisation of embryonic stem cell-derived mice by coat colour and permit the recovery of pure inbred mice from test crosses with C57BL6/N mice. The team therefore designed a targeting strategy to delete the retrotransposon from the locus and restore agouti gene function.
When they injected three of these ‘restored’ clones (JM8A) into albino blastocysts, all three produced chimeras with a high percentage agouti coat colour contribution and germline colonisation, with one clone, JM8A3, showing particularly favourable results. Since these JM8A lines are heterozygous for the corrected agouti allele, testcrosses with C57BL/6N mice yield embryonic stem cell–derived offspring with either agouti or black coats.
Finally, to assess the suitability of these JM8A3 cells for high-throughput gene targeting, the team performed targeting experiments and measured the clonal transmission rate of targeted clones as above. They found a clonal germline transmission rate of 80% from the injection of 11 targeted clones.
All of the cell lines produced in this project are available on request from the Knock Out Mouse Project repository and the European Conditional Mouse Mutagenesis repository.