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Rapid microinjection of fertilized eggs.
The chapter focuses on methods for using a picospritzer to inject large numbers of eggs that have been immobilized in open plastic dishes. The microinjection of reagents into eggs and early embryos aids in analyzing many cellular and developmental processes and is essential in studying cell lineages and cell fate specification, gene regulation, gene function, and biochemical pathways. This approach is especially useful for rapid, repetitive microinjection of reagents, such as morpholino antisense oligonucleotides, mRNAs, and DNA constructs. Many other molecules and cellular components can also be introduced into oocytes and zygotes by microinjection, including sperm, cytoplasm, calcium, aequorin, phosphatases, peptides, and antibodies. The advantages of pressure-based microinjection as a delivery method are that it allows precise control over (1) the amount of reagent introduced into the cell, (2) the time of introduction, and (3) the specific cell(s) into which the reagent is introduced. Current methods for microinjecting the oocytes and embryos of echinoderms and other invertebrate deuterostomes are based on a pressure-injection method. The chapter describes specific methods currently used in the laboratory for microinjecting the fertilized eggs ofStrongylocentrotus purpuratus and Lytechinus variegatus, two species of sea urchins. With slight modifications, the same methods are also useful for microinjecting molecules into specific blastomeres of early cleavage stage embryos.