High-Throughput Production of Gene Replacement Mutants in Neurospora crassa
| Autor: | Liubov Litvinkova, Susan Curilla, Liande Li, Christopher M. Crew, Wei Wang, Katherine A. Borkovich, Svetlana Krystofova, Norma Gorrochotegui-Escalante, Jay C. Dunlap, Lorena Altamirano, Hildur V. Colot, Patrick D. Collopy, Gyungsoon Park, Carol S. Ringelberg |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Recombination
Genetic Genetics Fungal protein Neurospora crassa biology Genes Fungal Mutant Crassa Genomics biology.organism_classification Article Fungal Proteins Blotting Southern Electroporation Transformation Genetic Cloning Molecular Genome Fungal Homologous recombination Gene Functional genomics Gene Deletion Gene knockout |
| Zdroj: | Methods in Molecular Biology ISBN: 9781617790393 |
| Popis: | The model filamentous fungus Neurospora crassa has been the focus of functional genomics studies for the past several years. A high-throughput gene knockout procedure has been developed and used to generate mutants for more than two-thirds of the ~10,000 annotated N. crassa genes. Yeast recombinational cloning was incorporated as an efficient procedure to produce all knockout cassettes. N. crassa strains with the Δmus-51 or Δmus-52 deletion mutations were used as transformation recipients in order to reduce the incidence of ectopic integration and increase homologous recombination of knockout cassettes into the genome. A 96-well format was used for many steps of the procedure, including fungal transformation, isolation of homokaryons, and verification of mutants. In addition, development of software programs for primer design and restriction enzyme selection facilitated the high-throughput aspects of the overall protocol. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|