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Oosightâ„¢-Enabled Enucleation for Somatic Cell Nuclear Transfer
Sponsored,vendor-submitted protocol   Sponsored by Cambridge Research & Instrumentation, Inc. (CRi)    Published in BioTechniques Protocol Guide 2007 (p.19)

Background

Somatic Cell Nuclear Transfer (SCNT) is the process of removing from an unfertilized oocyte its own chromosomes (enucleation) and replacing them with chromosomes taken from a somatic cell. Parthenogenesis may then be induced in order to create an embryonic clone of the donor of the somatic DNA, with either the intent of producing a live-birth clone, or of producing embryonic stem cells compatible with the somatic cell donor. The process of cloning is highly inefficient because each of the many sequential steps is inefficient, including the step of SCNT. We describe the use of the CRi Oosight™ system and the contribution it makes by increasing the efficiency of the vital process of SCNT. Visualization for Enucleation

In general, when viewing an oocyte one cannot see the chromosomes or the spindle on which they are held. Therefore, practitioners usually choose either to remove the chromosomes blindly, or to use fluorescent contrast agents and a UV light-source to reveal them. The former approach can leave the chromosomes in place or can result in the removal of too much cytoplasm. The latter has been successful in some species, but the cytotoxicity of UV light has proven harmful in others, including many primates. For these reasons, the efficiency of SCNT could be considerably improved by a new, reliable, non-toxic visualization method. The CRi Oosight System

The CRi Oosight imaging system is a development of the older LC-PolScope™ and SpindleView™ imaging systems. It enables real-time visualization of biological structures containing significant molecular order, such as the zona pellucida and the spindle. These features are revealed simultaneously on the computer-screen and through the microscope eyepieces. Visualization is reliable and requires no dangerous exogenous agents or UV light (indeed, Oosight and SpindleView systems are in use in many fertility clinics worldwide). The Oosight system is compatible with all common, modern, research-grade microscopes and contrast techniques and its use requires little modification of existing enucleation procedures.


Figure 1. (Click to enlarge)


Oosight-Enabled Enucleation

Healthy oocytes that have matured to the point of readiness for enucleation and that are maintained at a temperature congruous with their well-being can be brought, in glass-bottomed dishes, to a microscope fitted with an Oosight system and viewed immediately. The practitioner adjusts focus and oocyte orientation and can then obtain a clear view of the spindle. Upon seeing the spindle, the practitioner will then re-orient the oocyte for enucleation, typically positioning the spindle in the oocyte's equatorial plane, opposite the holding pipette. Enucleation can then proceed by use of either a piezo drill or a sharp, beveled pipette. No cytotoxic UV light is needed, no guess-work is required, a minimum of cytoplasm is removed and enucleation can be confirmed by observing the removed spindle.

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