IMPROVED METHOD FOR HOMOLOGOUS RECOMBINATION IN FUNGAL CELLS

摘要

The present invention discloses a method a method to construct fungal cells having a target sequence in a chromosomal DNA sequence replaced by a desired replacement sequence, comprising: providing a DNA molecule comprising a first DNA fragment comprising a desired replacement sequence flanked at its 5' and 3' sides by DNA sequences substantially homologous to sequences of the chromosomal DNA flanking the target sequence and a second DNA fragment comprising an expression cassette comprising a gene encoding diphtheria toxin A and regulatory sequences functional in the fungal cell operable linked thereto; transforming the fungal cells with the resulting DNA molecule; growing the cells to obtain transformed progeny cells having the DNA molecule inserted into the chromosome, wherein cells in which the DNA molecule is inserted in the chromosome via a non-homologous recombination event are selectively killed by expression of diphtheria toxin A; and obtaining cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence.