piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids.
We describe an efficient method for generating new piggyBac insertions in the germline of F(1) hybrid Tribolium castaneum derived from crosses between transgenic helper and donor strains. Helper strains carried single Minos elements encoding piggyBac transposase. The donor strain carried a single piggyBac element inserted into an actin gene, expanding the eye-specific, 3xP3-EGFP (enhanced green fluorescent protein) reporter expression domain to include muscle. Remobilization of the donor element is accompanied by loss of muscle fluorescence but retention of eye fluorescence. In a pilot screen, the piggyBac donor was remobilized in 84% of the hybrid crosses, generating hundreds of new lethal, enhancer-trap, semisterile and other insertions. The jumpstarter system described herein makes genome-wide, saturation insertional mutagenesis a realistic goal in this coleopteran species.
Lorenzen
M D
MD
Division of Biology, Kansas State University, Manhattan, KS, USA.
Kimzey
T
T
Shippy
T D
TD
Brown
S J
SJ
Denell
R E
RE
Beeman
R W
RW
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003433:Crosses, Genetic; D018983:DNA Footprinting; D004251:DNA Transposable Elements / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010641:Phenotype; D017422:Sequence Analysis, DNA; D019895:Transposases; D014231:Tribolium / Q000235:genetics*
D000199:Actins; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; C473499:Minos transposase; D019895:Transposases