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Production of cloned goats by nuclear transfer of cumulus cells and long‐term cultured fetal fibroblast cells into abattoir‐derived oocytes
Author(s) -
Lan GuoCheng,
Chang ZhongLe,
Luo MingJiu,
Jiang YunLiang,
Han Dong,
Wu YanGuang,
Han ZhengBin,
Ma SuoFeng,
Tan JingHe
Publication year - 2006
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20443
Subject(s) - biology , somatic cell nuclear transfer , andrology , blastomere , somatic cell , ionomycin , blastocyst , embryo , microbiology and biotechnology , transfection , cloning (programming) , fibroblast , cell culture , immunology , genetics , embryogenesis , gene , in vitro , medicine , computer science , programming language
Abstract Dairy goats are ideal for the transgenic production of therapeutic recombinant proteins. The use of recombinant somatic cell lines for nuclear transfer (NT) allows the introduction of genes by transfection, increases the efficiency of transgenic animal production to 100%, and overcomes the problem of founder mosaicism. Although viable animals have been cloned via NT from somatic cells of 11 species, the efficiency has been extremely low. Both blastomere and somatic cell NT increased fetal loss and perinatal morbidity/mortality in cattle and sheep, but fetal loss and perinatal mortality appear to be relatively low in goats. In this study, we produced cloned goats by NT from cumulus cells and long‐term cultured fetal fibroblast cells (FFCs) to abattoir‐derived oocytes. NT embryos were constructed from electrofusion of cumulus cells (CCs), FFCs, or skin fibroblast cells (SFCs) with cytoplasts prepared from abattoir‐derived ovaries. The NT embryos were activated with an optimized activating protocol (1 min exposure to 2.5 µM ionomycin followed by 2 hr incubation in 2mM 6‐DMAP). Two viable cloned kids from CCs and one from long‐term cultured FFCs (at passage 20–25) were born. Microsatellite analysis of 10 markers confirmed that all cloned offspring were derived from corresponding donor cells. To our knowledge, the production of cloned goat offspring using abattoir‐derived oocytes receiving nuclei from CCs and long‐term cultured FFCs has not been reported. The production of viable cloned animals after activation with reduced intensity of ionomycin and 6‐DMAP treatment has also not been reported. Loss of cloned embryos was obvious after 45 and 90 days of pregnancy, and a lack of cotyledons, heart defects, and improperly closed abdominal wall were observed in the aborted fetuses and one cloned kid. The fusibility and in vitro developmental potential of embryos reconstructed from FFCs at passage 20–25 were significantly lower than those of embryos reconstructed from FFCs at passage 3–5, and the cloning efficiency of the long‐term cultured cells was low (0.5%). Mol. Reprod. Dev. © 2006 Wiley‐Liss, Inc.