Tagging bloodmeals with phagemids allows feeding of multiple-sample arrays to single cages of mosquitoes (Diptera: Culicidae) and the recovery of single recombinant antibody fragment genes from individual insects.
A recombinant single-chain variable-region human antibody fragment (scFv) was expressed in Escherichia coli, extracted in hypertonic sucrose, mixed directly with blood and fed to Anopheles gambiae Giles mosquitoes. When E. coli containing the phagemids that encode these scFv were included in bloodmeals, phagemids could be recovered from the mosquito midgut for up to 3 d after feeding. Furthermore, large arrays of such gene-tagged scFv-containing bloodmeals could be fed to cages of mosquitoes using microtiter plates. Arrays of phagemids with and without an antibody insert were fed to single cages of mosquitoes to test whether individual mosquitoes fed from single wells of such arrays. Phagemids were recovered from 95% of blood-fed females and > 80% of these phagemids were monoclonal. Therefore, it is possible to feed multiple sample arrays of recombinant proteins to single cages of mosquitoes and to recover the genetic material that encodes for only one of the array elements from individual mosquitoes. This demonstration indicates that multiple-sample feeding and recovery strategies are feasible and may represent a viable strategy for future rapid screening of biologically active genes, gene products or microorganisms in live arthropods.
Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University Medical Center, New Orleans, LA 70112, USA.
G F
GF
Killeen
B D
BD
Foy
M
M
Shahabuddin
W
W
Roake
A
A
Williams
T J
TJ
Vaughan
J C
JC
Beier
D000818:Animals; D000852:Anopheles; D001769:Blood; D005247:Feeding Behavior; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D005456:Fluorescent Dyes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D016650:Fluorescein-5-isothiocyanate