Stable transformation of mosquito cell lines using a hsp70::neo fusion gene.
Mosquito cell culture transfection will allow the advancement of genetic studies of these important disease-transmitting insects. Towards this end, we report the generation of stably transformed Aedes aegypti Mos20 cells using a plasmid construct containing the Tn5 neo gene, the Drosophila melanogaster hsp70 promoter, an SV40 intron and poly adenylation sequence, and a pBR 322 backbone. The apparent frequency of transfection, as measured by transient resistance of cell colonies to Geneticin (G418), ranged between 1 x 10(-4) and 1 x 10(-5), whereas the mean frequency of transformation, as assessed by establishment of cloned lines, was 3.3 x 10(-6). The stable cell lines display typical characteristics common to mammalian cell lines transformed with plasmids, including stable resistance to G418 after removal of selection, and co-transformation with unlinked plasmids. However, in contrast to the report of transformation of Ae. albopictus cells [Monroe et al., Proc. Natl. Acad. Sci. USA 89 (1992) 5725-5729], the plasmids within transformed Ae. aegypti cells have a wide range of copy number (3 to 5000), are extensively rearranged, and are only found integrated into the chromosome.
Lycett
G J
GJ
Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, UK.
Crampton
J M
JM
D000330:Aedes / Q000166:cytology / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D004274:DNA, Recombinant / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D019868:Kanamycin Kinase; D008745:Methylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; C063842:dnaK protein, E coli