Handmade somatic cell cloning in cattle: Analysis of factors contributing to high efficiency in vitro

• Published in: Biology of reproduction 2003-02, Vol.68 (2), p.571-578
• Main Authors: Vajta, G, Lewis, I.M, Trounson, A.O, Purup, S, Maddox-Hyttel, P, Schmidt, M, Pedersen, H.G, Greve, T, Callesen, H
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Biotechnology; blastocyst; Blastocyst - ultrastructure; Blood Physiological Phenomena; blood serum; bovine serum albumin; cattle; Cattle - blood; Cattle - embryology; cell differentiation; cell fusion; cloning (cells); Cloning, Organism - methods; Culture Media; Early stages. Segmentation. Gastrulation. Neurulation; embryo (animal); embryo culture; embryo reconstruction; Embryo Transfer; embryogenesis; Embryology: invertebrates and vertebrates. Teratology; Embryonic and Fetal Development; Female; fetal calf serum; Fundamental and applied biological sciences. Psychology; Gene transfer; Genetic engineering; Genetic technics; in vitro embryo production; In Vitro Techniques; Methods. Procedures. Technologies; Micromanipulation; Microscopy, Electron; mitogenic activity; Mitogens; Nuclear Transfer Techniques; nuclear transplantation; Pregnancy; Pregnancy Rate; Synthetic digonucleotides and genes. Sequencing; ultrastructure
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod.102.008771

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.