Agouti C57BL/6N embryonic stem cells for mouse genetic resources

Abstract

We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background.

Figure 1

Properties and origin of Agouti JM8 C57BL/6N embryonic stem cells. (a) JM8 parental embryonic stem cells cultured on (top) SNL feeder cells in medium containing 15% serum and (bottom) gelatin-coated plates in medium supplemented with 10% serum and LIF. Scale bar: 100 μm (b) Genotype of embryonic stem cells and mice using a panel of 19 SNPs that differentiate between the N and J substrains of C57BL/6 (see Supplementary Table). JM8 embryonic stem cells were homozygous for all N alleles (shown in blue), confirming their origin from the C57BL/6N substrain. (c) Schematic of targeting strategy to restore Agouti function in JM8 cells. Exons (black boxes), retrotransposon (blue box), neo-TK selection cassette (N). For full details see Methods online. (d) F₁ offspring from a male chimera, generated by injection of JM8A3 cells into a C57BL/6-Tyr^c-Brd blastocyst, crossed to a C57BL/6-Tyr^c-Brd female. Both mice shown are embryonic stem cell-derived and the mouse carrying the corrected allele (A^tm1Brd, bottom) has an agouti coat. (e) CGH plot showing no copy number variants in JM8A3 cells compared to JM8.F6 cells. Relative copy number is plotted as the log ratio of hybridization signals of probes from JM8A3 DNA compared to JM8.F6. The JM8.F6 line has no copy number variants relative to C57BL/6NTac mice (see Supplementary Fig. 1).

Figure 2

Coat color of chimeric mice and their offspring from injections of JM8 and JM8A3 cells into blastocysts from several common mouse strains. Possible test crosses to detect germline contribution are indicated, along with the expected coat color(s) and genetic composition of embryonic stem cell-derived (ESC-derived) and host-derived G₁ offspring. For a full explanation of chimera coat colors see Supplementary Note online.