Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP

被引:94
作者
Handler, AM [1 ]
Harrell, RA [1 ]
机构
[1] USDA ARS, Ctr Med Agr & Vet Entomol, Gainesville, FL 32608 USA
关键词
piggyBac vector; germ-Line transformation; green fluorescent protein; position effect suppression; Anastrepha suspensa; tephritidae;
D O I
10.1016/S0965-1748(00)00119-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Germ-line transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac Vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species. Published by Elsevier Science Ltd.
引用
收藏
页码:199 / 205
页数:7
相关论文
共 20 条
[1]   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].
Altschul, SF ;
Madden, TL ;
Schaffer, AA ;
Zhang, JH ;
Zhang, Z ;
Miller, W ;
Lipman, DJ .
NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402
[2]   Genetic techniques - A universal marker for transgenic insects [J].
Berghammer, AJ ;
Klingler, M ;
Wimmer, EA .
NATURE, 1999, 402 (6760) :370-371
[3]  
Cary L.C., 1989, Virology, V161, P8
[4]   GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION [J].
CHALFIE, M ;
TU, Y ;
EUSKIRCHEN, G ;
WARD, WW ;
PRASHER, DC .
SCIENCE, 1994, 263 (5148) :802-805
[5]  
Coates C. J., 1998, P NATL ACAD SCI USA, V95, P3742
[6]   FACS-optimized mutants of the green fluorescent protein (GFP) [J].
Cormack, BP ;
Valdivia, RH ;
Falkow, S .
GENE, 1996, 173 (01) :33-38
[7]   Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti [J].
Cornel, AJ ;
Benedict, MQ ;
Rafferty, CS ;
Howells, AJ ;
Collins, FH .
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1997, 27 (12) :993-997
[8]   A NUCLEAR GFP THAT MARKS NUCLEI IN LIVING DROSOPHILA EMBRYOS - MATERNAL SUPPLY OVERCOMES A DELAY IN THE APPEARANCE OF ZYGOTIC FLUORESCENCE [J].
DAVIS, I ;
GIRDHAM, CH ;
OFARRELL, PH .
DEVELOPMENTAL BIOLOGY, 1995, 170 (02) :726-729
[9]   Germline transformation of Drosophila melanogaster with the piggyBac transposon vector [J].
Handler, AM ;
Harrell, RA .
INSECT MOLECULAR BIOLOGY, 1999, 8 (04) :449-457
[10]   The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly [J].
Handler, AM ;
McCombs, SD ;
Fraser, MJ ;
Saul, SH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1998, 95 (13) :7520-7525