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Microinjection of Helobdella (leech) embryos.

Authors: David A, Weisblat; Dian-Han, Kuo;

Microinjection of Helobdella (leech) embryos.

Abstract

One advantage of using Helobdella (leech) embryos as an experimental system is their amenability for microinjection. Blastomeres ranging in size from the zygote (400 microm diameter) down to micromeres and primary blast cells (approximately 20 microm diameter) can be injected by pressure under a dissecting microscope. Smaller cells can be injected by iontophoresis under a compound microscope. Microinjection is useful for studying embryonic development. For example, developmental fates of a cell can be followed by injecting a lineage tracer. A specific cell can be killed by injecting a toxic substance. Furthermore, cells can be killed at a given developmental stage by directing intense fluorescence illumination or a blue laser beam on fluorescein-labeled cells. Finally, gene expression can be manipulated in leech embryos by injecting zygotes or selected blastomeres with synthetic mRNA, morpholino antisense oligo, or a plasmid construct. Molecules <1500 Da can diffuse freely among early blastomeres via gap junctions. When intercellular diffusion of an injected substance is undesirable, small molecules should be conjugated to larger molecules such as dextran (10 kDa). Different commercial microinjection setups can be adopted for Helobdella embryos. This article describes how to microinject embryos using a versatile homemade pressure injection system. Under a dissecting microscope, embryos are immobilized by suction in a custom-fabricated chamber with the target cell facing upward. Cells are visualized using transillumination via a long-working-distance, dark-field condenser. The tip of a micropipette is brought into the target cell with a micromanipulator, and the injectant is delivered into the cell by pressure.

Related Organizations
Keywords

Blastomeres, Embryo, Nonmammalian, Microinjections, Gap Junctions, Equipment Design, Iontophoresis, Oligonucleotides, Antisense, Microscopy, Fluorescence, Leeches, Pressure, Animals, RNA, Messenger, Developmental Biology, Plasmids

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Powered by OpenAIRE graph
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
2
Average
Average
Average
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