efficient method to optimize Kluyveromyces lactis gene targeting

Kluyveromyces lactis strains impaired in the nonhomologous end-joining pathway are relevant tools for the homologous integration of exogenous DNA into the genome, as in the mutant strains, close to 100% of the integrants are targeted to the homologous locus, compared with a few per cent for the wild...

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Bibliographic Details
Published in:FEMS yeast research 2011-09, Vol.11 (6), p.509-513
Main Author: Wésolowski-Louvel, Micheline
Format: Article
Language:English
Subjects:
Cre‐loxP system
Deoxyribonucleic acid
DNA
Gene disruption
gene replacement
Gene targeting
Gene Targeting - methods
Genetics, Microbial - methods
genome
Genomes
homologous recombination
Kluyveromyces - genetics
Kluyveromyces lactis
Kluyveromyces marxianus var. lactis
loci
Mutagenesis, Insertional - methods
Mutants
NEJ1
NHEJ
Nucleotide sequence
plasmids
Recombinase
Recombination, Genetic
Yeast
yeasts
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Description
Summary:Kluyveromyces lactis strains impaired in the nonhomologous end-joining pathway are relevant tools for the homologous integration of exogenous DNA into the genome, as in the mutant strains, close to 100% of the integrants are targeted to the homologous locus, compared with a few per cent for the wild-type recipient. Using a loxP-kanMX-loxP cassette together with a Cre-recombinase plasmid, a nej1∷loxP mutant strain suitable for multiple gene disruption has been constructed. Furthermore, using this strain, PCR-generated constructs with only 50 bp of homologous flanking sequences resulted in efficient exogenous DNA targeting.
ISSN:1567-1356
1567-1364
DOI:10.1111/j.1567-1364.2011.00741.x