Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development
Figure 2
Analysis of Gga2 null mice (SYA176 strain).
A) Insertion of the gene-trap cassette within intron-1 of the mouse Gga2 (SYA176) gene results in normal GGA2 mRNA being disrupted by the splice acceptor site of the engrailed-2 leader sequence fusing with codon-33 (encoding L33) of the mouse Gga2 gene. The progeny of Gga2+/− inter-crosses were genotyped by subjecting cDNA to RT-PCR with Gga2 primer sets fwd1/rev1 (spanning exons-1 through 5) and fwd1/rev2 (spanning exon-1 and the β-galactosidase exon) (Figure S1A), which differentiated between the wt and null genotypes, respectively. B) RT-PCR of cDNA prepared from white blood cells or brain tissue was performed using Gga2 specific primers (reactions i) or a Gga2 specific primer in combination with a gene trap specific primer (reactions ii). PCR band in reactions (i) indicates wt genotype, while PCR band in reactions (ii) indicates null genotype. C) Determination of the insertion site of the gene-trap within intron-1 of the mouse Gga2 gene (SYA176) allowed for the design of Gga2 primer sets fwd1/rev1 and fwd1/rev2 (Figure S1A), which clearly distinguished between wt and null genotypes, respectively, at the gene level. D) PCR of genomic DNA was performed using Gga2 specific primers (reactions i) or a Gga2 specific primer in combination with a gene trap specific primer (reactions ii). PCR band in reactions (i) indicates wt genotype, while PCR band in reactions (ii) indicates null genotype. E) The genotypes of the Gga2+/− crosses were determined either by RT-PCR (day1 pups) or Western blots (embryos E9, E10, E11 and E12). No homozygous null were obtained out of 162 newborns or embryos that were analyzed. F) PCR was performed using genomic DNA from newborn pups of SYA176 Gga2+/− crosses in the C57BL/6J background. No homozygous nulls were obtained out of 45 pups analyzed.