Osmani Aysha H, Oakley Berl R, Osmani Stephen A
Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA.
Nat Protoc. 2006;1(5):2517-26. doi: 10.1038/nprot.2006.406.
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.
在异核体拯救技术中，利用pyrG营养标记在构巢曲霉中通过同源重组进行基因缺失，以取代靶基因的编码区。如果一个必需基因被缺失，无效等位基因会在自发产生的异核体中得以维持，这些异核体由两种基因不同类型的细胞核组成。一种核类型的必需基因被缺失，但具有功能性的pyrG等位基因（pyrG+）。另一种具有必需基因的野生型等位基因，但缺乏功能性的pyrG等位基因（pyrG-）。因此，对由初级转化体形成的单核无性孢子进行简单的生长测试，可以将非必需基因的缺失与必需基因的缺失区分开来，后者只能通过异核体拯救进行繁殖。生长测试还能够确定无效等位基因的表型。诊断性PCR可用于在分子水平上确认缺失。该技术适用于大规模的基因缺失计划，并且可以在3周内完成。
