Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Poster Abstracts 205
endothelial specific promoter active in pigs and thus target expression
of hA20 to the endothelial cell layer. Providing the endothelial cells
with a higher antiapoptotic potential could decrease their
susceptibility to cell death.
P537
Expression of an omega-3 fatty acid desaturase gene
from scarlet flax in bovine transgenic embryos cloned
from transfected somatic cells
Saeki, K 1 *, Indo, Y 1 , Tatemizo, A 1 , Suzuki, I 2 , Matsumoto, K 1 , Hosoi, Y 1 ,
Murata, N 3
1Department of Genetic Engineering, School of Biology-Oriented Science and
Technology, Kinki University, Japan; 2 Laboratory of Plant Physiology and
Metabolism, University of Tsukuba, Japan; 3 National Institute for Basic
Biology, Japan
Introduction Long chain n-3 fatty acids are considered desirable in
human diets because they can lower the risk of coronary artery
disease, cancer and inflammatory diseases. n-3 fatty acids are found in
fish oils and specific plant oils. But their levels in animal meats are
quite low, because mammals lack the gene for converting linoleic acid
to alfa-linolenic acid. Recently, it has been reported that a humanized
nematode gene, hfat-1, can be used to increase the levels of n-3 fatty
acids in transgenic pigs. We report here functional expression of a
plant-derived gene for an omega-3 fatty acid desaturase (FAD3) in
gene-transfected bovine cells and production of embryos cloned from
the transfected cells.
Methods The gene was isolated from immature seeds of scarlet flax,
because the level of alfa-linolenic acid of the flax seeds is the highest
among terrestorial plants. Bovine muscle satellite cells were isolated
from a 9-month-old male calf. The codon usage of the flax FAD3
cDNA was optimized (humanized) for high expression in mammalian
cells. A plasmid (pIRES2-EGFP) containing the humanized FAD3
gene (hFAD3) under the control of the CAG promoter, and a
neomycin-resistance cassette (pCAG/hFAD3/IRES/EGFP/(neor)) was
transfected to the satellite cells with a transfection reagent (
GeneJammer ). The stably transfected cells were differentiated to
multilocular adipocytes by culturing with bFGF, dexamethasone and
octanoic acid. Total lipids were isolated from the adipocytes and their
fatty acid composition was analyzed by gas chromatography. We then
produced cloned bovine embryos using the hFAD3 cells. The cloned
embryos were cultured to the blastocyst stage. Blastocyst rates and
gene expression of EGFP and hFAD3 were then examined.
Results The level of total n-3 fatty acids in the hFAD3 cells (12.5%)
was higher than that in the control cells (9.1%, P4 to 8 mm in diameter) sizes of ovarian
follicles were transfected with liposome alone, both liposome and
EGFP, or liposome and EGFP in combination with pEGISI
respectively. The transfection procedure was performed according to
the Lipofectamine2000 manufacturers’ instructions. Cell proliferation
was quantified by the CellTiter 96® AQueous One Solution Cell
Proliferation Assay. Cell apoptosis was detected using an Annexin V-
FITC/Propidium Iodide for flow cytometry. Steroidogenesis was
evaluated by measurements of both estradiol and progesterone in
culture via radioimmunoassay methods. Maturation of oocytes cocultured
with transfected GCs and subsequent embryo developments
were also investigated.
Results The transfection with pEGISI inhibited proliferation of GCs
from medium follicles (P