title: An alternative method for delivering exogenous material into developing zebrafish embryos.
abstract: Non-invasive manipulation of multicellular systems is important for medical and biological research. The ability to introduce, remove, or modify molecules in the intracellular environment is pivotal to our understanding of cellular structure and function. Herein, we report on an alternative method for introducing foreign material into developing embryos using the application of femtosecond (fs) laser pulses. When intense fs laser pulses are focused to a sub-micron spot, transient pores are formed, providing a transport pathway for the delivery of exogenous material into embryonic cells. In this study, zebrafish embryos were used as a model system to demonstrate the non-invasiveness of this applied delivery tool. Utilizing optically induced transient pores chorionated and dechorionated zebrafish embryos were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate), Streptavidin-conjugated quantum dots or DNA (Simian-CMV-EGFP). Pore formation was independent of the targeted location, with both blastomere-yolk interface and blastomere pores competent for delivery. Long-term survival of laser manipulated embryos to pec-fin stage was 89% and 100% for dechorionated and chorionated embryos, respectively. To our knowledge, this is the first report of DNA delivery into zebrafish embryos utilizing fs laser pulses.
authors.affiliation: Ultrafast Photonics and Nano-Optics Laboratory, Centre for Nanoelectronics, Nanophotonics & Nanoscale Systems, Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada. vkohli@ece.ualberta.ca
authors.forename: Vikram
authors.initials: V
authors.lastname: Kohli
authors.forename: Vanesa
authors.initials: V
authors.lastname: Robles
authors.forename: M Leonor
authors.initials: ML
authors.lastname: Cancela
authors.forename: Jason P
authors.initials: JP
authors.lastname: Acker
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Waskiewicz
authors.forename: Abdulhakem Y
authors.initials: AY
authors.lastname: Elezzabi
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D017874:Immediate-Early Proteins / Q000235:genetics; D042541:Intracellular Space / Q000378:metabolism; D007834:Lasers; D008297:Male; D036103:Nanotechnology / Q000379:methods; D010539:Permeability; D045663:Quantum Dots; D014162:Transfection / Q000379:methods; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D005456:Fluorescent Dyes; D017874:Immediate-Early Proteins; C044582:immediate-early proteins, cytomegalovirus; D016650:Fluorescein-5-isothiocyanate