Protocol abstract
Nature Protocols 2, - 296 - 304 (2007)
Published online: 1 March 2007 | doi:10.1038/nprot.2007.7
Subject Categories: Cell and tissue culture | Model organisms
Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)
Naoko Yoshida1 & Anthony CF Perry1
Abstract
The mouse is a genetically tractable model organism widely used to study mammalian development and disease. However, mouse metaphase II (mII) oocytes are exquisitely sensitive and intracytoplasmic sperm injection (ICSI) with conventional pipettes generally kills them. This problem can be solved with piezo-actuated micromanipulation, in which the piezo-electric effect (crystal deformation in response to an externally applied voltage) propels a microinjection needle tip forward in a precise and rapid movement. Piezo-actuated micromanipulation enhances the penetration of membranes and matrices, and mouse ICSI is a major application. Here we describe a comprehensive, step-by-step mouse piezo ICSI protocol for non-specialists that can be completed in 2–4 h. The protocol is a basic prelude to multiple applications, including nuclear transfer cloning, spermatid injection, blastocyst injection, mII transgenesis, and streamlining micromanipulation in primates and livestock. Moreover, piezo ICSI can be used to obtain offspring from 'dead' (non-motile) sperm, enabling trivial sperm freezing protocols for mouse strain storage and shipment.
- Laboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Correspondence to: Anthony CF Perry1 e-mail: tony@cdb.riken.jp
nature-products
A-Z product listing
- 5-ml polypropylene bottles(Perfector Scientific)
- Borosilicate glass capillaries(Sutter)
- BSA(Sigma)
- Dumostar #5 fine biological forceps(Electron Microscopy Sciences)
- Embryo-tested bovine testis hyaluronidase(Sigma)
- Falcon 10-cm (100
20 mm) Optilux petri dishes(Becton Dickinson)
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