Fig. 9

Molecular analysis of the trapped gene mamdc2a in transgenic zebrafish.

(A) Schematic representation of the insertion of gene trap cassette in the intron7 of gene. The locations of primers used for molecular analysis are indicated. (B) Integrity analysis of gene trap cassette in transgenic fish. Genomic DNA was extracted from the trail of transgenic fish and subjected to PCR analysis with primes indicated in (A). (C) RT-PCR analysis of mamdc2a transcript in wild-type (+/+), heterozygous (+/-) and homozygous (-/-). Primers E7F/E8R were used to amplify the endogenous transcript and primers E7F/R3 were used to amplify the exon7-EGFP fusion transcript. The β-actin transcript was amplified as an internal control. (D) The fusion transcript generated from the proper splicing of EGFP gene and its upstream exon in mamdc2a gene. Sequences in red represent exon7 of mamdc2a gene. Sequences in yellow represent the SA signal. Sequences in gray indicate the IRES followed by the partial EGFP sequence in green. (E) qRT-PCR was performed with primers E7F/E8R to determine the expression of endogenous mamdc2a gene in wild-type, heterozygous and homozygous embryos at 72 hpf. The mamdc2a levels were normalized to the β-actin mRNA levels. (F) qRT-PCR analysis of SB11 expression in 24 hpf embryos from mamdc2a-transgenic line after heat shock at 37°C for 3 h and recovery at 28°C for 2 h. (G) Detection of Cre-mediated TiHsp-SB11 excision by southern hybridization. Cre reombinase mRNA was microinjected into wild-type, heterozygous and homozygous embryos. “+” represents the presence of Cre recombinase and “-” represents the absence of Cre recombinase. Line1–2: DNA from wild-type. Line 3–4: DNA from heterozygous. Line 5–6: DNA from homozygous.