Abstract
Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.
Highlights
Manipulation of gene function in vivo is an indispensable technique for modifying individual functions of targeted cells in living animals
We examined the usefulness of our photoporation technique in a range of vertebrate embryos (Figure 3) by introducing the larger molecule (10,000-Da dextran) into single neurons of embryos from a model animal and a non-model animal
In this paper, we have shown for the first time that biomolecules can be introduced into individual cells of diverse live nontransgenic embryos using the presented femtosecond laser photoporation technique
Summary
Manipulation of gene function in vivo is an indispensable technique for modifying individual functions of targeted cells in living animals. Transgene expression in a transgenic worm was successfully manipulated by using a heat shock promoter and focusing a continuous-wave infrared laser on a single cell in the transgenic animal [1] This system is only applicable to transgenic animals carrying a gene driven by the heat shock promoter. We injected a cocktail of mini-ruby dextran and a DNA plasmid (pCAGGS-EGFP) between the vitelline membrane and the epithelium of stage 14–16 chick embryos and irradiated single cells with 400-nJ laser pulses. We examined the usefulness of our photoporation technique in a range of vertebrate embryos (Figure 3) by introducing the larger molecule (10,000-Da dextran) into single neurons of embryos from a model animal (mouse) and a non-model animal (shark). At 24 h post-injection, dextran was successfully delivered to single neurons of cultured mouse embryos (Fig. 3B). From the four animals tested, it stands to reason that our photoporation technique could be applied to a wide range of vertebrate embryos
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