A new variant of self-excising ß-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa.
J Microbiol Methods
; 100: 17-23, 2014 May.
Article
em En
| MEDLINE
| ID: mdl-24556286
ABSTRACT
In a previous study, we developed a cassette employing a bacterial ß-recombinase acting on six recognition sequences (ß-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette. A tedious subsequent procedure was needed to purify homokaryons due to the lack of a negative selection after cassette eviction. Additionally, the endoxylanase xylP promoter from Penicillium chrysogenum used in the construct was not strongly regulated in N. crassa, which led to low efficiency in cassette eviction. Herein we report an improved variant of the self-excising ß-recombinase/six cassette for repetitive gene deletions in N. crassa using a native endoxylanase gh10-2 promoter from N. crassa, plus the introduction of a bidirectional selection marker to facilitate homokaryon selection using a thymidine kinase (tk) gene (negative selection) in addition to the phosphinothricin resistance gene (bar(r)) (positive selection).
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Mutagênese Insercional
/
Recombinases
/
Técnicas de Inativação de Genes
/
Genética Microbiana
/
Neurospora crassa
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article