Expression of a Synthetic Gene of CTDM by Transgenic Animals.
BACKGROUND
The purpose of this study was to produce molecules that can precisely regulate the complement and coagulation system and to assess the expression of such molecules in transgenic animals.


METHODS
The CTDM gene, which is composed of the delta-1-99 amino acid (aa) C1-INH, EGF domain 4-6 of thrombomoduline (TM), short consensus repeat (SCR) 2-4 of DAF(CD55), and SCR 2-4 of MCP(CD46) was established. The codon usage for expression in mammals was adopted. The cDNA of CTDM was subcloned into the pCPI site (the human insulin promoter and a cytomegalovirus enhancer). pCPI-CTDM was transfected into pig endothelial cells (PEC). The expression of the molecule was clearly assessed by means of flow cytometry.


RESULTS
BD3F1 female mice were induced to superovulate and were then crossed with BD3F1 males. Micro-injection and embryo transfer were performed by standard methods, thus generating transgenic mice that express CTDM. The mice carried the CTDM plasmid, as verified by PCR. Tissue expression levels in transgenic mouse lines generated with the constructs were follows: pancreas, 1.0; brain, 5.4; thymus, 0.3; heart, 0.2; lung, 1.2; liver, 0.1; kidney, 0.1; intestine, 0.4; and spleen, 1.6. A naive control mouse was also analyzed in the exact manner as for the transgenic mice.


CONCLUSIONS
A synthetic CTDM gene with codon usage optimized to the mammalian system represents a critical factor in the development of transgenic animals.
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Electronic address: u377126a@ecs.osaka-u.ac.jp.
R
R
Sakai
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
A
A
Maeda
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
R
R
Matsuura
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
H
H
Eguchi
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
P
P
Lo
Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
H
H
Hasuwa
Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
M
M
Ikawa
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
K
K
Nakahata
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
M
M
Zenitani
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
T
T
Yamamichi
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
S
S
Umeda
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
H
H
Okuyama
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
S
S
Miyagawa
D000818:Animals; D001777:Blood Coagulation / Q000235:genetics*; D018958:CD55 Antigens / Q000235:genetics; D003001:Cloning, Molecular; D003165:Complement System Proteins / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005813:Genes, Synthetic / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D051916:Membrane Cofactor Protein / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D013552:Swine; D018180:Thrombomodulin / Q000235:genetics; D014162:Transfection / Q000379:methods; D014183:Transplantation, Heterologous
D018958:CD55 Antigens; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D051916:Membrane Cofactor Protein; D018180:Thrombomodulin; D003165:Complement System Proteins