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. 2014 Jan 12;23(12):3085–3101. doi: 10.1093/hmg/ddu013

Figure 1.

Figure 1.

Conditional disruption of Tll1 and Bmp1. (A) Structure of targeting vector and Tll1 locus before and after homologous recombination and subsequent Flp- and Cre-induced recombination to produce alleles Tll1loxP and Tll1Δ6, respectively. Arrows indicate directionality for transcription of neor (yellow box). Open box, TK selection gene. Green boxes represent Tll1 exons. Frt and loxP sequences are indicated by blue and red arrowheads, respectively. P, PstI. Orange and blue boxes represent 1.1-kb Eco72I-NheI and 496-bp HpaI-PstI 5′ and 3′ external Southern blot probes, respectively. (B) Genomic Southern blot analysis of PstI-restricted DNA from wild-type (WT) or correctly targeted (Tll1+/loxP−frt) ES clones. The 5′ probe detected 13.2-kb and 6.9-kb bands from wild-type and targeted alleles, respectively. The 3′ probe detected the 13.2-kb wild-type allele band, and a 5.3-kb band corresponding to the targeted allele. (C) Excision of the flrted neomycin resistance cassette. PCR genotyping of tail genomic DNA from mice heterozygous for the Tll1loxP−frt-targeted allele, and from mice heterozygous for the Tll1loxp allele, from which the flrted neor cassette has been excised via crossing with the ACTB:FLPe line of Flp ‘deleter’ mice. Bands of 663, 770 and 1130 bp correspond to wild type, Tll1loxP and Tll1loxP−frt alleles, respectively. (D) RT–PCR of total RNA from femora shows tamoxifen-induced excision of floxed Tll1 sequences. Bands of 179 and 544 bp correspond to TllΔ6 (excised) and Tllflox (unexcised control) alleles, respectively. (E) Green boxes and red arrowheads represent Bmp1 exons and LoxP sites, respectively, in floxed Bmp1 allele. (F) Screening of tamoxifen-induced excision of floxed alleles using PCR genotyping of ear genomic DNA shows extent of tamoxifen-induced excision of floxed Bmp1 sequences from a BTKO mouse. Bands of 233 and 298 bp correspond to Bmp1flox (unexcised) and Bmp1Δ3−6 (excised) alleles, respectively. BTKO mice with <75% excision were excluded from further study. (G) RT–PCR of total RNA from femora shows tamoxifen-induced excision of floxed Bmp1 sequences. Bands of 243 and 811 bp correspond to Bmp1Δ3−6 (excised) and Bmp1flox (unexcised control) alleles, respectively.