Agouti C57BL/6N embryonic stem cells for mouse genetic resources
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1 nature methods Agouti N embryonic stem cells for mouse genetic resources Stephen J Pettitt, Qi Liang, Xin Y Rairdan, Jennifer L Moran, Haydn M Prosser, David R Beier, Kent Lloyd, Allan Bradley & William C Skarnes Supplementary figures and text: Supplementary Figure 1 Supplementary Figure 2 Supplementary Table 1 Supplementary Note Comparative genome hybridization of the JM8.F6 and JM8.N4 sublines. Fixing the agouti locus. SNP alleles and genotyping results. Chimera coat colors observed when using JM8 Agouti embryonic stem cells. Note: Supplementary Data 1 and 2 are available on the Nature Methods website.
2 Supplementary Figure 1 Comparative genome hybridization of the JM8.F6 and JM8.N4 sublines. (a) JM8.F6 embryonic stem cell vs. NTac mouse genomic DNA shows no copy number variation at a 0.2 Mb resolution. (b) JM8.N4 embryonic stem cell vs. JM8.F6 embryonic stem cell genomic DNA reveals a copy number gain of 1.7 Mb on distal Chr. 10.
3 Supplementary Figure 2 Fixing the agouti locus. (a) Targeting scheme to repair the nonagouti mutation. The nonagouti (a) insertion is shaded in light grey, regions homologous to the targeting vector (ATV-20) are indicated by a thick line, exons by numbered vertical black bars (not to scale). The differences between the wild-type A allele and the targeted allele (A tm1brd ) close to the insertion site are shown at the bottom of this panel. PCR primers used in C are indicated by single headed arrows and the probe used in B by a black rectangle. (b) Southern blot (SphI digest) using probe shown in A for genotyping the initial replacement. 1 - correctly targeted 2 - incorrectly targeted (nonagouti band only) (c) PCR using primers indicated in A for genotyping selection cassette removal after FLPe treatment and confirming transmission of allele to F 1. Lanes: 1, molecular weight markers; 2-4, FIAU resistant targeted clones (lanes two (JM8A1) and four (JM8A3) have lost the selection cassette); 5, 129S7 embryonic stem cells (Agouti); 6, N mouse ear; 7-9, F 1 ears; 10, no template. (d) Ventral views of a mouse carrying the spontaneous reverted A W-J allele 20 (left) and a mouse carrying the targeted reverted allele reported here (right). The targeted allele confers an all-over agouti coat, with only a slight lightening on the ventrum, whereas the spontaneous allele results in a cream-colored ventrum (white-bellied agouti phenotype).
4 Supplementary Table 1 SNP alleles and genotyping results position (bp; NCBI SNP Chr m37) J JCrl Tyr c-brd NTac SI6 JM8 rs G A A A A A CEL-2_ A A A C A C rs G G G C G C rs T T T C T C rs T T T C T C rs T T T A T A rs A A G G A G rs T C T C C C rs T T T C T C rs G G G A G A rs T T T C T C rs A A A T A T rs G G G A G A rs G G G T G T rs G G G A G A rs A C A C C C rs A A G G A G rs A A A G A G rs T T T C T C
5 Supplementary Note Chimera coat colors observed when using JM8 Agouti embryonic stem cells. The Agouti gene encodes a secreted signaling peptide, and therefore acts in a non cell-autonomous manner. Transient expression of Agouti is required in cells surrounding the pigment-producing melanocytes of the hair follicle to produce an agouti hair (mostly black with a subapical yellow band). By contrast, the tyrosinase gene (Tyr), mutation of which is responsible for the albino phenotype, acts cell autonomously with respect to the follicle melanocytes. All embryonic stem cells described here are Tyr +, thus contribution of embryonic stem cells to the hair follicle melanocytes is sufficient to produce a pigmented hair. When using embryonic stem cells bearing a functional Agouti gene injected into a nonagouti albino -Tyr c host, the exact color observed, i.e. black or agouti, depends on the extent of embryonic stem cell contribution to the cells surrounding the follicle melanocytes. If chimeras have only limited embryonic stem cell contribution, there may be insufficient Agoutiexpressing cells to produce an agouti hair and the animals may appear black and white rather than agouti and white. The situation can vary for different patches of hair, depending on the extent of embryonic stem cell contribution in the particular region. These animals will still produce agouti pups if the embryonic stem cells have contributed to the germ line. Another non cell-autonomous effect of Agouti is seen when using BALB/c blastocysts for injection. In this case all host cells express Agouti, but not Tyr. From injections of wild-type JM8 (nonagouti) embryonic stem cells, some of the normally black embryonic stem cell-derived hairs will be agouti, provided the embryonic stem cell-derived melanocytes in the follicle are surrounded by enough Agouti expressing cells derived from the host blastocyst.
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Europe PMC Funders Group Author Manuscript Published in final edited form as: Nat Methods. 2009 July ; 6(7): 493 495. doi:10.1038/nmeth.1342. Agouti C57BL/6N embryonic stem cells for mouse genetic resources
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