Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique

In the heterokaryon rescue technique, gene deletions are carried out using the pyrG nutritional marker to replace the coding region of target genes via homologous recombination in Aspergillus nidulans. If an essential gene is deleted, the null allele is maintained in spontaneously generated heterokaryons that consist of two genetically distinct types of nuclei. One nuclear type has the essential gene deleted but has a functional pyrG allele (pyrG(+)). The other has the wild-type allele of the essential gene but lacks a functional pyrG allele (pyrG(-)). Thus, a simple growth test applied to the uninucleate asexual spores formed from primary transformants can identify deletions of genes that are non-essential from those that are essential and can only be propagated by heterokaryon rescue. The growth tests also enable the phenotype of the null allele to be defined. Diagnostic PCR can be used to confirm deletions at the molecular level. This technique is suitable for large-scale gene-deletion programs and can be completed within 3 weeks.