Figure 1
Figure 1. Deletion of Flip in myeloid linage results in postnatal growth retardation and premature death. (A) Schematic of the Flip targeting strategy. A 9.5-kb Flip genomic DNA segment (exon 2 to 4 from a C57BL/6J mouse BAC library) was used to engineer the Flip conditional mutagenesis construct. Exons are indicated by rectangular boxes and E and B represent the restriction cleavage sites of EcoRI and BamHI. Three loxP sites were inserted as indicated by triangles. The dashed lines represent the flanking region for Southern blot probes. The location of 3 PCR primers (a, b, c) and orientation are indicated by arrows. Amplification using primers a and b generates a 220-bp fragment for wild-type (Flip+) and a 270-bp fragment for the floxed (Flipf) allele. Cre-induced recombination generates a 150-bp fragment for depleted Flip allele (Flipd) using primers a and c. All recombinant DNA and animal procedures were approved by the Office of Research Safety and the Institutional Animal Care and Use Committee of Northwestern University. (B) PCR genotyping of the LysM-cre–induced cell type-specific deletion of Flipf. The representative Flipf/+, LysMc/+ littermate control and Flipf/d, LysMc/+ knockout (KO) mice were genotyped from tail biopsy, and the different cell types were isolated from bone marrow (BM), and peripheral blood were genotyped by 3 PCR primers indicated in panel A. Granulocytes (Gran) were 11b+/Gr1+ F4/80−; peripheral blood monocytes or bone marrow monocyte precursors (Mono) were 11b+, F4/80+; B cells were 11b−/CD19+, and T cells were 11b−CD3+. (C) Body weight of Flipf/d, LysMc/+ (n = 53) and littermate controls including: Flip+/+, Flipf/+ or Flipf/f, LysM+/+ (n = 9); Flipf/d or Flip+/d, LysM+/+ (n = 10); Flip+/+, LysMc/+ (n = 5); Flipf/+or Flip+/d, LysMc/+ (n = 24); and Flip+/+, Flipf/+or Flip+/d, LysMc/c (n = 12). All mice are between 6 to 24 weeks of age. ***P < .001, compared with indicated groups. (D) Postnatal viability of Flipf/d, LysMc/+ (n = 101) and the Flipf/d, LysMc/c (n = 17) and littermate controls (n = 475), which included: Flip+/+, Flipf/+ or Flipf/f, LysM+/+ (n = 112), Flipf/d or Flip+/d, LysM+/+ (n = 112), Flip+/+, LysMc/+ (n = 47), Flipf/+or Flip+/d, LysMc/+ (n = 127), and Flip+/+, Flipf/+or Flip+/d, LysMc/c (n = 77) mice.

Deletion of Flip in myeloid linage results in postnatal growth retardation and premature death. (A) Schematic of the Flip targeting strategy. A 9.5-kb Flip genomic DNA segment (exon 2 to 4 from a C57BL/6J mouse BAC library) was used to engineer the Flip conditional mutagenesis construct. Exons are indicated by rectangular boxes and E and B represent the restriction cleavage sites of EcoRI and BamHI. Three loxP sites were inserted as indicated by triangles. The dashed lines represent the flanking region for Southern blot probes. The location of 3 PCR primers (a, b, c) and orientation are indicated by arrows. Amplification using primers a and b generates a 220-bp fragment for wild-type (Flip+) and a 270-bp fragment for the floxed (Flipf) allele. Cre-induced recombination generates a 150-bp fragment for depleted Flip allele (Flipd) using primers a and c. All recombinant DNA and animal procedures were approved by the Office of Research Safety and the Institutional Animal Care and Use Committee of Northwestern University. (B) PCR genotyping of the LysM-cre–induced cell type-specific deletion of Flipf. The representative Flipf/+, LysMc/+ littermate control and Flipf/d, LysMc/+ knockout (KO) mice were genotyped from tail biopsy, and the different cell types were isolated from bone marrow (BM), and peripheral blood were genotyped by 3 PCR primers indicated in panel A. Granulocytes (Gran) were 11b+/Gr1+ F4/80−; peripheral blood monocytes or bone marrow monocyte precursors (Mono) were 11b+, F4/80+; B cells were 11b−/CD19+, and T cells were 11b−CD3+. (C) Body weight of Flipf/d, LysMc/+ (n = 53) and littermate controls including: Flip+/+, Flipf/+ or Flipf/f, LysM+/+ (n = 9); Flipf/d or Flip+/d, LysM+/+ (n = 10); Flip+/+, LysMc/+ (n = 5); Flipf/+or Flip+/d, LysMc/+ (n = 24); and Flip+/+, Flipf/+or Flip+/d, LysMc/c (n = 12). All mice are between 6 to 24 weeks of age. ***P < .001, compared with indicated groups. (D) Postnatal viability of Flipf/d, LysMc/+ (n = 101) and the Flipf/d, LysMc/c (n = 17) and littermate controls (n = 475), which included: Flip+/+, Flipf/+ or Flipf/f, LysM+/+ (n = 112), Flipf/d or Flip+/d, LysM+/+ (n = 112), Flip+/+, LysMc/+ (n = 47), Flipf/+or Flip+/d, LysMc/+ (n = 127), and Flip+/+, Flipf/+or Flip+/d, LysMc/c (n = 77) mice.

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