Document Detail


Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.
MedLine Citation:
PMID:  12533421     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Gábor Vajta; Ian M Lewis; Alan O Trounson; Stig Purup; Poul Maddox-Hyttel; Mette Schmidt; Hanne Gervi Pedersen; Torben Greve; Henrik Callesen
Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biology of reproduction     Volume:  68     ISSN:  0006-3363     ISO Abbreviation:  Biol. Reprod.     Publication Date:  2003 Feb 
Date Detail:
Created Date:  2003-01-20     Completed Date:  2003-08-01     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0207224     Medline TA:  Biol Reprod     Country:  United States    
Other Details:
Languages:  eng     Pagination:  571-8     Citation Subset:  IM    
Affiliation:
Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark. gabor.vajta@agrsci.dk
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MeSH Terms
Descriptor/Qualifier:
Animals
Blastocyst / ultrastructure
Blood Physiological Phenomena
Cattle* / blood,  embryology
Cloning, Organism / methods*
Culture Media
Embryo Transfer
Embryonic and Fetal Development
Female
Micromanipulation
Microscopy, Electron
Mitogens
Nuclear Transfer Techniques
Pregnancy
Pregnancy Rate
Chemical
Reg. No./Substance:
0/Culture Media; 0/Mitogens

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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