Eppendorf Piezo-actuated Mouse ICSI (intracytoplasmic sperm Bedienungsanleitung PDF herunterladen

Hadi Hajarian, Laboratory of In Vitro Fertilization, Agro-Biotechnology Institute (ABI), Malaysia

Ko Kwan Mor, Eppendorf Asia Pacific Headquarters, Malaysia

No 037 | September 2011 - revised August 2012

Userguide

ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted reproductive technology in

both, humans and animals. The Mouse is one of the most common model organisms of choice to study mammalian

fertilization. However, the ability to fertilize mouse eggs successfully by sperm injection has been hard to achieve due

to the fact that the metaphase II mouse oocytes are extremely sensitive and convential ICSI gives low survival rates

[1]. This problem can be solved with piezo-actuated micromanipulation where the capillary advances a very short

distance at a very high speed. This enables the capillary to penetrate the cell membrane with minimum distortion of

the cell and yields to high survival rates. The microinjection workstation required for this technique is very similar to

standard ICSI, but with the addition of a piezo-assisted unit attached to the capillary holder. In this Userguide, the

use of the Eppendorf PiezoXpert in combination with the Eppendorf TransferMan

NK 2 workstation is shown and

parameter settings as well as optimization of the piezo-actuated microinjection procedure itself are discussed.

Abstract

While this conventional ICSI technique has been very suc-

cessful in humans, it has proven unsuccessful in mice [2].

This is due to a lower viscosity of the ooplasm. Thus wound

healing capacity of mouse oocytes is inferior to that of hu-

man oocytes. Furthermore, the oolemma of mouse oocytes

is much more elastic than that of human oocytes. Suc-

cessful ICSI in mice was first demonstrated by Kimura and

Yanagimachi [3] using piezo-actuated micromanipulation,

which is far less traumatic than the conventional method.

This method proved to increase survival as well as fertiliza-

tion rates of oocytes after sperm injection [3].

Eppendorf has a long tradition in the area of conventional

ICSI. In particular, the Eppendorf TransferMan NK 2 system

is an electronic micromanipulation system that offers a num-

ber of useful features for ICSI. In combination with the piezo

impact unit PiezoXpert, Eppendorf is offering a complete

system for both, conventional ICSI and piezo-actuated ICSI

(Figure 1, 2).

Introduction

Piezo-actuated Mouse ICSI (intracytoplasmic

sperm injection) using the Eppendorf PiezoXpert®

Fig. 1: Actuator of PiezoXpert mounted onto the right arm of

the manipulator (Eppendorf TransferMan NK 2).

Intracytoplasmic sperm injection (ICSI) is a technique that

involves the direct transfer of a single sperm into the oocyte

cytoplam via a glass capillary with a spike.

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Inhaltsverzeichnis

Seite 1 - Userguide

Hadi Hajarian, Laboratory of In Vitro Fertilization, Agro-Biotechnology Institute (ABI), MalaysiaKo Kwan Mor, Eppendorf Asia Pacific Headquarters, Mal

Seite 2 - Userguide No 037

Animals, media, consumables and devices were used as described previously [4] with the following modifications:Media: CZB-HEPES (CZB-H) CZB CZB-HEPES

Seite 3 - Fig. 4:

Sperm dropletSperm head droplet Userguide No 037 | Page 34. 1 Installation of PiezoXpert onto TransferMan NK 2Loosen the M3x12 cheese head screw

Seite 4

Userguide No 037 | Page 4Figure 7: Preparation of microinjection capillary. Loosely screw the grip head into the front knurled screw of the ac

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Userguide No 037 | Page 5programs can be stored. Each program consists of param-eter set A and B. Usually, parameter set A is used for the penetration

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Userguide No 037 | Page 6Fig. 10: Mouse ICSIMove the stage to the injection droplet. Place the holding capillary using the previous stored positions.

Seite 7 - Userguide No 037

[1] Yoshida N and Perry ACF. Piezo-actuated Mouse Intracytoplasmic Sperm Injection (ICSI). Nature Protocols 2007; Vol. 2, No. 2:296-304. [2] Colla

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Userguide NoYour local distributor: www.eppendorf.com/worldwideEppendorf AG · 22331 Hamburg · Germany · Tel: +49 40 53801-0 · Fax: +49 40 538 01-556 ·