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  • Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.

Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.

Methods in molecular biology (Clifton, N.J.) (2012-06-20)
Mauricio Niklitschek, Marcelo Baeza, María Fernández-Lobato, Víctor Cifuentes
ABSTRACT

Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker. To accomplish this, in a first step, a heterozygous hygromycin B-resistant strain is obtained by a single process of transformation (carrying the inserted hph gene). Following, the heterozygous mutant is grown in media with increasing concentrations of the antibiotic. In this way, the strains that became homozygous (by mitotic recombination) for the antibiotic marker would able to growth at higher concentration of the antibiotic than the heterozygous. The method can be potentially applied for obtaining double mutants of other diploid organisms.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hygromycin B solution from Streptomyces hygroscopicus, ≥60% (HPLC), 45-60 mg/mL in H2O
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, suitable for plant cell culture, BioReagent, ≥60% (HPLC), lyophilized powder
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, lyophilized powder
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, powder, BioReagent, suitable for cell culture, suitable for insect cell culture