title: Hand-made cloning approach: potentials and limitations.
abstract: Two major drawbacks hamper the advancement of somatic cell nuclear transfer in domestic animals. The first is a biological problem that has been studied extensively by many scientists and from many viewpoints, including the cell, molecular and developmental biology, morphology, biochemistry and tissue culture. The second is a technical problem that may be responsible for 50% or more of quantitative and/or qualitative failures of routine cloning experiments and is partially the result of the demanding and complicated procedure. However, even the relatively rare documented efforts focusing on technique are usually restricted to details and accept the principles of the micromanipulator-based approach, with its inherent limitations. Over the past decade, a small alternative group of procedures, called hand-made cloning (HMC), has emerged that has the common feature of removal of the zona pellucida prior to enucleation and fusion, resulting in a limited (or no) requirement for micromanipulators. The benefits of HMC are low equipment costs, a simple and rapid procedure and an in vitro efficiency comparable with or higher than that of traditional nuclear transfer. Embryos created by the zona-free techniques can be cryopreserved and, although data are still sparse, are capable of establishing pregnancies and resulting in the birth of calves. Hand-made cloning may also open the way to partial or full automation of somatic cell nuclear transfer. Consequently, the zona- and micromanipulator-free approach may become a useful alternative to traditional cloning, either in special situations or generally for the standardisation and widespread application of somatic cell nuclear transfer.
authors.affiliation: Section of Reproductive Biology, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark. gabor.vajta@agrsci.dk
authors.forename: G
authors.initials: G
authors.lastname: Vajta
authors.forename: P M
authors.initials: PM
authors.lastname: Kragh
authors.forename: N R
authors.initials: NR
authors.lastname: Mtango
authors.forename: H
authors.initials: H
authors.lastname: Callesen
mesh_terms: D000818:Animals; D000829:Animals, Domestic / Q000235:genetics; D002417:Cattle / Q000196:embryology; D019976:Cloning, Organism / Q000379:methods*; D015925:Cryopreservation / Q000662:veterinary; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000648:ultrastructure*; D012756:Sheep / Q000196:embryology; D013552:Swine / Q000196:embryology; D015044:Zona Pellucida / Q000502:physiology