Expression of a Synthetic Gene of CTDM by Transgenic Animals

• Published in: Transplantation proceedings Vol. 48; no. 4; pp. 1279 - 1281
• Main Authors: Sakai, R, Maeda, A, Matsuura, R, Eguchi, H, Lo, P, Hasuwa, H, Ikawa, M, Nakahata, K, Zenitani, M, Yamamichi, T, Umeda, S, Okuyama, H, Miyagawa, S
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2016
• Subjects: Animals; Blood Coagulation - genetics; CD55 Antigens - genetics; Cloning, Molecular; Complement System Proteins - genetics; DNA, Complementary - genetics; Endothelial Cells - metabolism; Female; Flow Cytometry; Genes, Synthetic - genetics; Homeodomain Proteins - genetics; Humans; Male; Membrane Cofactor Protein - genetics; Mice; Mice, Transgenic; Promoter Regions, Genetic; Surgery; Swine; Thrombomodulin - genetics; Transfection - methods; Transplantation, Heterologous
• ISSN: 0041-1345; 1873-2623
• DOI: 10.1016/j.transproceed.2015.10.067

Abstract 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.