In vivo transfection of developmentally competent Brugia malayi infective larvae.
Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70 kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.
Global Health Infectious Disease Research Program, Department of Global Health, University of South Florida, Tampa, FL 33612, USA.
Shulin
S
Xu
Canhui
C
Liu
George
G
Tzertzinis
Elodie
E
Ghedin
Christopher C
CC
Evans
Ray
R
Kaplan
Thomas R
TR
Unnasch
D000818:Animals; D019470:Biolistics; D017178:Brugia malayi / Q000235:genetics* / Q000254:growth & development* / Q000472:pathogenicity; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008842:Microfilariae / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000379:methods*
D018509:DNA, Helminth; D012333:RNA, Messenger; D008156:Luciferases