Fig. 3. Generating the Hand-null mutant by homologous recombination. (A) Strategy for replacing the Hand-coding region with the mini-white gene by ends-out homologous recombination. (B) RT-PCR of the four mutant lines obtained from the screen showing no detectable Hand transcript in homozygous mutant larvae. (C) In situ hybridization using Hand probe (green) did not detect any Hand transcripts in homozygous Hand mutants (lower panel), which were distinguished from heterozygous Hand mutant embryos (upper panel) by using anti-ß-Gal antibody (red) to detect the presence of the balancer chromosome.