Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell.
The present study was conducted to establish an efficient production system for bovine transgenic somatic cell nuclear transfer (SCNT) embryos, the effect of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos were examined with their expression rates of a marker gene. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6, as a carrier. In Experiment 1, three types of bovine cells were transfected at passages 2 to 4, and then trypsinized and GFP-expressing cells were randomly selected and used for SCNT. Developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In all cell types used, GFP expression rates of SCNT embryos gradually decreased with the progression of embryo development. In Experiment 2, the effect of passage number of cumulus cells in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed, but significantly higher GFP expression was shown in blastocysts reconstructed with cumulus cells at early passage. In Experiment 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 microm) or small cell (<30 microm)] at passages 2 to 4 were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells as well as fetal cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.
Cho
Jongki
J
Department of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea.
Bhuiyan
M M U
MM
Shin
Sangtae
S
Park
Eulsoon
E
Jang
Goo
G
Kang
Sungkeun
S
Lee
Byeongchun
B
Hwang
Woosuk
W
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000196:embryology*; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D004622:Embryo, Mammalian / Q000196:embryology*; D005347:Fibroblasts / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase