Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.
We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.
Tamura
T
T
National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan.
Thibert
C
C
Royer
C
C
Kanda
T
T
Abraham
E
E
Kamba
M
M
Komoto
N
N
Thomas
J L
JL
Mauchamp
B
B
Chavancy
G
G
Shirk
P
P
Fraser
M
M
Prudhomme
J C
JC
Couble
P
P
Toshiki
T
T
Chantal
T
T
Corinne
R
R
Toshio
K
K
Eappen
A
A
Mari
K
K
Natuo
K
K
Jean-Luc
T
T
Bernard
M
M
Gérard
C
C
Paul
S
S
Malcolm
F
F
Jean-Claude
P
P
Pierre
C
C
D000199:Actins / Q000235:genetics; D000375:Aging / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D012831:Bombyx / Q000196:embryology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003433:Crosses, Genetic; D004251:DNA Transposable Elements / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D018095:Germ-Line Mutation / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007814:Larva / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008297:Male; D009036:Moths / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011679:Pupa / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D020079:Terminal Repeat Sequences / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics*; D019076:Transgenes / Q000235:genetics; D019895:Transposases / Q000235:genetics / Q000378:metabolism
D000199:Actins; D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019895:Transposases