Targeted disruption of the mouse

Csf1r gene: decreased growth rate and increased circulating CSF-1 inCsf1r⁻/Csf1r⁻mice. (A) The targeted region of the Csf1r gene, theCsf1r gene-targeting vector, and the correctly targeted allele, showing exons 1 to 7, restriction enzyme sites (As,AseI; H, HindIII; A, ApaI; C,ClaI), PCR primers used (P1-P6, see text), the in-frame humanized green fluorescent protein (hGFP) sequence and the neomycin resistance (PGKneo) and thymidine kinase (PGKtk) cassettes above the flanking intron 7 probe were used to identify the 7-kb wild-type allele and 8-kb targeted allele HindIII fragments depicted below it. (B) Southern blot analyses of the DNA from individual G418 and gancyclovir-resistant ES cell clones using the probe shown in A. Asterisks mark clones possessing the correctly targeted allele. (C) Anti–CSF-1R Western blot analysis of BMM (upper panel). The molecular masses of the mature CSF-1R (165 kDa) and its precursor (130 kDa) are indicated. RT-PCR of BMM RNA with primers P5, P6 (A) specific for the targeted allele (middle panel) and control primers for β-actin (lower panel) are shown. (D) Growth curves of progeny of double heterozygote (Csf1r⁺/Csf1r⁻;Csf1⁺/Csf1^op×Csf1r⁺/Csf1r⁻;Csf1⁺/Csf1^op) crosses (n ≥ 5 for each genotype). (E) Serum CSF-1 concentration determined by a radioimmunoassay that selectively detects biologically active CSF-1 (± SD; n ≥ 5 for each genotype).