使用極體顯微鏡施行卵子去核及體細胞核植入

使用極體顯微鏡Polscope施行卵子去核及體細胞核植入SCNT

http://cshprotocols.cshlp.org/content/2008/9/pdb.prot5040.full

Noninvasive Human Nuclear Transfer with Embryonic Stem Cells

1. Sohyun L. McElroy1 and 
2. Renee A. Reijo Pera

+Author Affiliations

1. Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, CA 94304-5542, USA

1. ↵¹Corresponding author (sohyun@stanford.edu)

 

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INTRODUCTION

In somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is transferred to an enucleated oocyte for reprogramming to an embryonic cell state through the use of the endogenous machinery. SCNT technology has been used to produce offspring, establish embryonic stem cells, and study epigenetic reprogramming, as mediated by oocytes, in several animal species. In humans, there are ethical and practical issues that limit availability of oocytes donated by women of reproductive age specifically for research. Thus, there is a need to more exhaustively explore alternatives, including oocyte sources and different SCNT protocols. Nuclear transfer (NT) techniques are important factors that impact development of NT embryos. The procedures of enucleation of oocyte genetic material and introduction of the donor nucleus vary depending on species and laboratories. Hoechst staining has been used successfully for invasive enucleation in many animal studies, though it is known that Hoechst staining and ultraviolet (UV) light can damage oocyte mitochondrial DNA. More recently, noninvasive NT techniques that rely on polarized microscopic imaging systems have been used to visualize the meiotic spindle without DNA staining and UV illumination. This protocol describes a method for noninvasive human nuclear transfer by visualizing the oocyte spindle without DNA staining.