Figure 1
Figure 1. The hypomorphic Cbfbrss allele. (A) Targeting vector, targeted Cbfbrss(neo) locus, and the Cbfbrss allele following excision of floxed neo by Tg(CMV-Cre). The Cbfbrss allele contains the lacZ gene in the reverse orientation flanked by an extra copy of exon 4, inserted into intron 3. The targeting vector also contains a recombination signal sequence (RSS12) in intron 3, and the RSS23 sequence in intron 4. Cbfb exons are black, introduced coding sequences (lacZ, floxed-neo, TK) are gray, and both the loxP sequences flanking neo and the RSS sequences are indicated by triangles. The expected sizes of the XhoI restriction fragments that hybridize with the 3′ probe and the BamHI and XbaI fragments that hybridize with the 5′ probe are indicated. PCR primers for genotyping are indicated by arrows on the Cbfbrss allele. A indicates AvrII; B, BamHI; X, XhoI; S, SalI; and Xb, XbaI. (B) Southern blots showing correct targeting (prior to neo excision) at both the 5′ and 3′ ends. Top shows BamHI digest; bottom, XhoI digest. (C) RT-PCR showing the 2 predominant mRNAs generated from the Cbfbrss allele. The top band contains all 6 Cbfb exons and encodes a 22-kDa protein, while the bottom band lacks exon 4 and encodes a 17.5-kDa protein. RT-PCR for Hprt was used as a loading control. Arrows on the schematic diagram show the location of RT-PCR primers. (D) Western blot of lysates prepared from CD45+ cells isolated from 17.5-dpc FLs probed for actin (top) and CBFβ (bottom). Lysate from 4 × 104 Cbfb+/+ cells was loaded in lane 1. The amount of Cbfbrss/+, Cbfb+/−, Cbfbrss/rss, and Cbfbrss/− lysates loaded in lanes 3, 5, 7, and 9 was adjusted based on the actin signal. Lanes 2, 4, 6, 8, and 10 are 2-fold dilutions of each lysate. The 22-kDa endogenous CBFβ protein, and the purified heterodimerization domain CBFβ (141) used as a blotting control are indicated with arrows. The measured amount of CBFβ in each sample was normalized relative to actin, and is expressed relative to Cbfb+/+ cells (averaged from 2-4 experiments) below the lanes. The calculated amount is based on the observation that approximately one third of the mRNA produced from the Cbfbrss allele is properly spliced. Wild-type CD45+ cells contain 2.4 × 105 CBFβ molecules/cell as determined by comparison with dilutions of the CBFβ (141) standard (not shown).

The hypomorphicCbfbrssallele. (A) Targeting vector, targeted Cbfbrss(neo) locus, and the Cbfbrss allele following excision of floxed neo by Tg(CMV-Cre). The Cbfbrss allele contains the lacZ gene in the reverse orientation flanked by an extra copy of exon 4, inserted into intron 3. The targeting vector also contains a recombination signal sequence (RSS12) in intron 3, and the RSS23 sequence in intron 4. Cbfb exons are black, introduced coding sequences (lacZ, floxed-neo, TK) are gray, and both the loxP sequences flanking neo and the RSS sequences are indicated by triangles. The expected sizes of the XhoI restriction fragments that hybridize with the 3′ probe and the BamHI and XbaI fragments that hybridize with the 5′ probe are indicated. PCR primers for genotyping are indicated by arrows on the Cbfbrss allele. A indicates AvrII; B, BamHI; X, XhoI; S, SalI; and Xb, XbaI. (B) Southern blots showing correct targeting (prior to neo excision) at both the 5′ and 3′ ends. Top shows BamHI digest; bottom, XhoI digest. (C) RT-PCR showing the 2 predominant mRNAs generated from the Cbfbrss allele. The top band contains all 6 Cbfb exons and encodes a 22-kDa protein, while the bottom band lacks exon 4 and encodes a 17.5-kDa protein. RT-PCR for Hprt was used as a loading control. Arrows on the schematic diagram show the location of RT-PCR primers. (D) Western blot of lysates prepared from CD45+ cells isolated from 17.5-dpc FLs probed for actin (top) and CBFβ (bottom). Lysate from 4 × 104Cbfb+/+ cells was loaded in lane 1. The amount of Cbfbrss/+, Cbfb+/−, Cbfbrss/rss, and Cbfbrss/− lysates loaded in lanes 3, 5, 7, and 9 was adjusted based on the actin signal. Lanes 2, 4, 6, 8, and 10 are 2-fold dilutions of each lysate. The 22-kDa endogenous CBFβ protein, and the purified heterodimerization domain CBFβ (141) used as a blotting control are indicated with arrows. The measured amount of CBFβ in each sample was normalized relative to actin, and is expressed relative to Cbfb+/+ cells (averaged from 2-4 experiments) below the lanes. The calculated amount is based on the observation that approximately one third of the mRNA produced from the Cbfbrss allele is properly spliced. Wild-type CD45+ cells contain 2.4 × 105 CBFβ molecules/cell as determined by comparison with dilutions of the CBFβ (141) standard (not shown).

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