Non-invasive Enucleation in SCNT
The process of cloning is very inefficient, with yields of only a few percent or less. Somatic cell nuclear transfer (SCNT), which involves removing the nucleus from an unfertilized oocyte (enucleation) and replacing it with a nucleus taken from a somatic cell, is an important step in this process. Two parameters that are critical to improving SCNT success are: a) the mechanical precision with which the arrested meiotic spindle is extracted from the oocyte; and b) the quality of the host oocyte. Prior to CRi’s Oosight™ system, practitioners removed the nucleus blindly, by referencing the site of the first polar body as a guide, or by employing fluorescent contrast agents and UV light-sources which are harmful to cells.
The utility and safety of SCNT has been considerably improved through the use of the non-toxic visualization method provided by Oosight (Figure 1). The Oosight system increases SCNT success by improving the precision of enucleation and the ability to assess oocyte quality. It has also been used to visualize the spindle during enucleation in various mammalian species (see Figure 2), including human, monkey, bovine, porcine, goat, horse, rabbit, mouse and hamster. In fact, a recent breakthrough in the field of primate embryonic stem cell research was largely attributed to using Oosight versus conventional methods (Mitalipov 2007). The following movie shows enucleation being performed by Dr. Shoukhrat Mitalipov: Movie of Enucleation of Rhesus Monkey Oocyte.
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Figure 1. CRi Oosight images of a) monkey, b) bovine, c) rabbit, d) horse e) human and f) hamster oocytes, respectively. Metaphase II spindle indicated by red arrow. Monkey image courtesy Ng S.C., Chen N.Q. et al, NUS, Singapore. Rabbit image courtesy Shi der Shen et al, Gung Xi University, China. Bovine and hamster images courtesy D. Keefe et al, Marine Biological Laboratory, Woods Hole, MA. Human oocyte image courtesy R. Scott, RMA, Morristown, NJ.
Figure 2. Comparative images of the same human MII oocyte. Image on the left was taken with Hoffman modulation contrast optics, showing no contrast of the spindle. Image on the right was acquired using the Oosight system, where a nice barrel-shaped spindle is visible (indicated by the arrowhead).
