Review of Enucleation Methods and Procedures Used in Animal Cloning: State of the Art

Abstract

Enucleation of a recipient oocyte is a crucially important process for nuclear transfer efficiency. Several procedures have been developed and used in the production of nuclear transfer embryos. Although the use of excitable fluorochromes and ultraviolet (UV) light are commonly used for complete enucleation, they also pose the risk of damaging the maternal cytoplast. Telophase and chemically assisted enucleation have also been used for cloning, but the quality and quantity of the recipient cytoplasm varies with the procedure used. This paper reviews various methods used for enucleation, and discusses their benefits and limitations with respect to cloning efficiency.

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

“…These abnormalities have been related to the incorrect or insufficient competence of the enucleated oocytes (cytoplasts) to remove epigenetic marks of the transferred nucleus and to reprogram it correctly first into a totipotent embryonic state and later into various differentiated states during post-implantation development (Yang et al 2007). In this sense, oocyte enucleation is a crucial step in SCNT procedures, because an excessive loss of cytoplasm with important factors involved in nuclear reprogramming or embryonic development may irremediably affect the overall success of the SCNT procedure (Fulka et al 2004, Li et al 2004.…”

“…In efforts to make oocyte enucleation technically easier and less invasive to the oocytes and to improve the competence of the resultant cytoplasts for SCNT, several approaches and timings for the removal of chromosome have been tested (reviewed in Fulka et al (2004) and Li et al (2004)). Among them, chemically assisted (AE) and chemically induced (IE) enucleation protocols could represent alternative models to ME.…”

Effects of the Timing of Cumulus Cell Removal from Bovine Oocytes on Enucleation Rate and Subsequent Development after Somatic Cell Nuclear Transfer

“…The rate of successful enucleation by this method is reportedly lower than those of Hoechst staining and chemical treatment [2,3]. On the other hand, enucleation using UV irradiation after staining of the oocyte DNA with Hoechst dye is an efficient and reliable method, but exposure of the recipient oocytes to Hoechst dye and UV irradiation may have detrimental effects on their DNA and cytoplasmic organelles [1,4]. Enucleation of oocytes after treatment with chemicals (e.g., demecolcine [2]) is also an efficient method, but the oocytes may be exposed to potentially harmful chemical stresses [5].…”

“…58: 615-619, 2012) E nucleation of recipient oocytes is an essential process in the production of nuclear transfer (NT) embryos. Although the nuclei of mouse and rat oocytes can be observed and removed under an inverted microscope, it is difficult to observe the nuclei of bovine, porcine, rabbit, goat and sheep oocytes without DNA staining because of their high lipid contents [1]. Therefore, in bovine NT, enucleation has been attempted by several methods such as the blind technique, Hoechst staining combined with UV irradiation and the chemically assisted method [1].…”

“…Although the nuclei of mouse and rat oocytes can be observed and removed under an inverted microscope, it is difficult to observe the nuclei of bovine, porcine, rabbit, goat and sheep oocytes without DNA staining because of their high lipid contents [1]. Therefore, in bovine NT, enucleation has been attempted by several methods such as the blind technique, Hoechst staining combined with UV irradiation and the chemically assisted method [1]. In blind enucleation, the removal of metaphase II (MII) chromosomes is usually carried out blindly by aspirating or pushing out the cytoplasm underlying the first polar body (PB1).…”