Figure 1
Targeted mutation of the Gpc1 locus creates a null allele. (A) Targeting strategy. A construct was created in which loxP sites flank exon 1 of Gpc1 and FLP recognition target (FRT) sites flank negative (TK) and positive (neo) selection markers. After transfection into embryonic stem (ES) cells, Southern blotting, using a probe located outside the targeted region, permitted verification of targeted integration. Transient tranfection of targeted ES cells with a Cre expression plasmid was then used to remove Gpc1 exon 1 and selection markers, prior to generation of mice. (B) Identification of the Gpc1 wild-type (+) and mutant (-) allele by PCR in mice. (C) Immunoblotting of adult brain membrane fractions with an anti-Gpc1 antibody. The presence of a discrete band found only in samples pretreated with heparitinase (Hase) demonstrates that the immunoreactive molecule is a heparan sulfate proteoglycan (HSPG) core protein. Note the decreased band intensity in heterozygous animals, and complete absence of immunoreactivity in homozygotes. The small apparent difference in mobility and slight tilt of the wild-type band is an artifact of uneven electrophoresis. (D) Immunoblotting of adult brain membrane HSPG core proteins using 3G10 antibody. Notice that the loss of a band at the correct molecular weight for Gpc1 in mutant animals is not accompanied by a substantial, consistent change in the presence of other HSPG cores. Hase, Heparitinase; Case, Condroitinase ABC. (E-G) Whole-mount in situ hybridization for Gpc1 at E8.5. Genotypes are as indicated. Red arrowheads point to areas of high Gpc1 expression in the developing brain and branchial arches. Notice that Gpc1 expression is greatly reduced or absent in homozygous mutants. Levels in heterozygotes are intermediate. (H, I) Whole-mount in situ hybridization for Gpc1 in wild-type (H) and Gpc1-/- (I) embryos at E9.5. Staining has been deliberately overdeveloped to show the absence of signal in the mutant, suggesting that mutant Gpc1 mRNA is unstable. FB, forebrain; HB, hindbrain; MB, midbrain.