Target-selected mutant screen by TILLING in Drosophila.
The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the approximately 15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.
Winkler
Sylke
S
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Schwabedissen
Anja
A
Backasch
Dana
D
Bökel
Christian
C
Seidel
Claudia
C
Bönisch
Stefanie
S
Fürthauer
Maximilian
M
Kuhrs
Antje
A
Cobreros
Laura
L
Brand
Michael
M
González-Gaitán
Marcos
M
D000818:Animals; D001483:Base Sequence; D003433:Crosses, Genetic; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism; D015723:Gene Library; D017344:Genes, Insect / Q000235:genetics*; D016678:Genome; D020180:Heteroduplex Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004720:Endonucleases; C406922:CEL I nuclease