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Fig. S1. Generation of Sfrp1 and Sfrp2 mutant mice. (A) Targeting strategy of Sfrp1. A 5¢ 1.4- kb BamHI-SacII fragment and a 3¢ 5.3-kb EagI-NheI fragment were ligated into a lacZ KI cassette. The cassette comprised pNTR T-lacZ (containing an IRES, a nuclear-localized lacZ open reading frame and an SV40 polyA addition sequence) and PGKneobpAlox (a PGK promoter-driven neomycin-resistance gene plus a bovine growth hormone polyA addition sequence between two loxP sequences). A MC1DtpA-negative-selection marker cassette (DT) was added to the 5¢ homologous arm to enrich homologous recombinants (Yagi et al., 1993). The targeting vector was linearized at an XhoI site outside the region of homology. Linearized vector (25 µg) was electroporated into 107 AB1 ES cells, which were subsequently cultured in the presence of G418 (250 µg/ml) (McMahon and Bradley, 1990). A black triangle indicates the location and orientation of a loxP site. (B) Southern analysis of DNA from wild-type and Sfrp1KI ES cell clones. G418-resistant clones (648 clones) were initially screened by Asp718 digestion and hybridized with a unique external 5¢ probe. Subsequently, targeted clones were examined by BamHI digestion and hybridization with a 3¢ probe to verify correct targeting. Three correctly targeted clones were identified, two of which were capable of contributing to the mouse chimera germline. (Top) Asp718-digested DNA hybridized with the 5¢ probe. An 11.0-kb fragment indicates the wild-type allele, whereas an 8.0-kb fragment denotes the mutant allele. (Bottom) BamHI-digested DNA hybridized with the 3¢ probe. A 14.0-kb and an 11.0-kb fragment indicate the wild-type and the mutant allele, respectively. (C) Southern blot (top) and PCR (bottom) analyses of tail DNA from offspring obtained from the heterozygous intercross. (Top) Asp718-digested DNA hybridized with the 5¢ probe. (Bottom) The SacIIf (5¢-GATTGGTTAACTGCGCGGCTG-3¢)/SacIIr (5¢-GACTGGAAGCTCACGTAGTCG-3¢) primer set yielded a 510-bp wild-type-specific fragment by PCR. The SacIIf-IRESr2 (5¢-GGGCCCTCACATTGCCAAAAG-3¢) primer set amplified a 420-bp mutant-specific fragment. Positions of genotyping primers (SacIIf, SacIIr and IRESr2) are indicated in A. (D) Targeting strategy of Sfrp2. A 5¢ 2.0-kb Asp718-EcoRI fragment and a 3¢ 6.5-kb EcoRV-BamHI fragment were ligated into a PGKneobpAloxB-positive selection marker cassette [PGK promoter-driven neomycin resistance gene plus bovine growth hormone polyA addition sequence between two loxP sequences (Shimono and Behringer, 2003)]. A MC1DtpA-negative selection marker cassette was added onto the 3¢ homologous arm (Yagi et al., 1993). The targeting vector was linearized at an Asp718 site. A black triangle indicates the location and orientation of a loxP site. (E) Southern blot analysis of DNA from wild-type and Sfrp2 targeted cell clones. G418 resistant clones (249 clones) were initially screened by BamHI digestion and hybridization with a unique external 5¢ probe. Subsequently, targeted clones were examined by EcoRI digestion and hybridization with a 3¢ probe. Three correctly targeted clones were identified, one of which was capable of contributing to the mouse chimera germline. (Top) BamHI-digested DNA hybridized with the 5¢ probe. A 14.0-kb fragment indicates the wild-type allele, whereas a 6.0-kb fragment indicates the mutant allele. (Bottom) EcoRI-digested DNA hybridized with the 3¢ probe. A 12.0-kb and a 10.0-kb fragment designate the wild-type and the mutant allele, respectively. (F) Southern blot (top) and PCR (bottom) analyses of tail DNA from offspring obtained from the heterozygous intercross. (Top) BamHI-digested DNA hybridized with the 5¢ probe. (Bottom) The 2gf1 (5¢-CACGAGTAGTGAATACCTGAG-3¢)/2gr1 (5¢-GACACATTGTTGCTGCTTCCT-3¢) primer set yielded a 480-bp wild-type-specific fragment by PCR. The 2gr1-bpAf (5¢-GATCAATTCTCTAGAGCTCGC-3¢) primer set amplified a 350-bp mutant-specific fragment. Positions of primers (2gf1, 2gr and bpAf) used for genotyping are depicted in D. The ends of the 5¢ and 3¢ exon of the Sfrp1 and Sfrp2 genes are depicted based on database information. pBS, pBluescript KS–.
References
McMahon, A. P. and Bradley, A. (1990). The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073-1085.
Shimono, A. and Behringer, R. R. (2003). Angiomotin regulates visceral endoderm movements during mouse embryogenesis. Curr. Biol. 13, 613-617.
Yagi, T., Nada, S., Watanabe, N., Tamemoto, H., Kohmura, N., Ikawa, Y. and Aizawa, S. (1993). A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Anal. Biochem. 214, 77-86.
Fig. S2. Expression of Wnt3a and tail bud markers in Sfrp1 and Sfrp2 double mutant embryos at E8.5. (A-L) Expression levels of Wnt3a (A-C; white arrow), Wnt5a (D-F), Fgf17 (G-I) and Fgf8 (J-L; black arrow) were unaffected in Sfrp1–/–;Sfrp2–/– embryos. (J¢-L¢) Dorsal view of the embryos in J-L. (M-O) The distribution pattern of phosphorylated ERK protein (dpERK) was suggestive of the Fgf pathway in the tail bud of Sfrp1–/–;Sfrp2–/– embryos (O), as well as in control embryos (M,N). Arrowheads indicate the domain corresponding to the expression of Fgf; an arrow indicates the lower domain of dpERK activity immediately prior to newly generated somites (asterisk). Precise stage of embryo is indicated by somite number. Scale bar: 500 µm.
Fig. S3. Wnt3a and Fgf8 expression are reduced in Sfrp1–/–;Sfrp2–/– embryos at E9.5. (A-F) Brachyury (A-C) and Fgf17 (D-F) expression in control (A,B,D,E) and Sfrp1–/–;Sfrp2–/– (C,F) embryos. Note that the tail bud was generated in Sfrp1–/–;Sfrp2–/– embryos. (G-L) Fgf8 (G-I; arrow) and Wnt3a (J-L; carved arrows) expression diminished in Sfrp1–/–;Sfrp2–/– embryos (I,L) relative to that in control embryos (G,H,J,K). (M-O) Wnt5a expression in control (M,N) and Sfrp1–/–;Sfrp2–/– embryos (O). FL, forelimb; A, anterior; P, posterior. Scale bars: 500 µm in A-I; 500 µm in M-O.
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