Expression of a Synthetic Gene of CTDM by Transgenic Animals

R. Sakai, A. Maeda, R. Matsuura, H. Eguchi, P. Lo, Hidetoshi Hasuwa, M. Ikawa, K. Nakahata, M. Zenitani, T. Yamamichi, S. Umeda, H. Okuyama, S. Miyagawa

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Pages (from-to)1279-1281
Number of pages3
JournalTransplantation Proceedings
Volume48
Issue number4
DOIs
Publication statusPublished - 2016 May 1
Externally publishedYes

Fingerprint

Synthetic Genes
Genetically Modified Animals
Transgenic Mice
Codon
Embryo Transfer
Cytomegalovirus
Epidermal Growth Factor
Thymus Gland
Intestines
Pancreas
Mammals
Flow Cytometry
Plasmids
Swine
Spleen
Endothelial Cells
Complementary DNA
Insulin
Kidney
Amino Acids

ASJC Scopus subject areas

  • Surgery
  • Transplantation

Cite this

Sakai, R., Maeda, A., Matsuura, R., Eguchi, H., Lo, P., Hasuwa, H., ... Miyagawa, S. (2016). Expression of a Synthetic Gene of CTDM by Transgenic Animals. Transplantation Proceedings, 48(4), 1279-1281. https://doi.org/10.1016/j.transproceed.2015.10.067

Expression of a Synthetic Gene of CTDM by Transgenic Animals. / Sakai, R.; Maeda, A.; Matsuura, R.; Eguchi, H.; Lo, P.; Hasuwa, Hidetoshi; Ikawa, M.; Nakahata, K.; Zenitani, M.; Yamamichi, T.; Umeda, S.; Okuyama, H.; Miyagawa, S.

In: Transplantation Proceedings, Vol. 48, No. 4, 01.05.2016, p. 1279-1281.

Research output: Contribution to journalArticle

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 2016, 'Expression of a Synthetic Gene of CTDM by Transgenic Animals', Transplantation Proceedings, vol. 48, no. 4, pp. 1279-1281. https://doi.org/10.1016/j.transproceed.2015.10.067
Sakai, R. ; Maeda, A. ; Matsuura, R. ; Eguchi, H. ; Lo, P. ; Hasuwa, Hidetoshi ; Ikawa, M. ; Nakahata, K. ; Zenitani, M. ; Yamamichi, T. ; Umeda, S. ; Okuyama, H. ; Miyagawa, S. / Expression of a Synthetic Gene of CTDM by Transgenic Animals. In: Transplantation Proceedings. 2016 ; Vol. 48, No. 4. pp. 1279-1281.
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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.",
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AU - Sakai, R.

AU - Maeda, A.

AU - Matsuura, R.

AU - Eguchi, H.

AU - Lo, P.

AU - Hasuwa, Hidetoshi

AU - Ikawa, M.

AU - Nakahata, K.

AU - Zenitani, M.

AU - Yamamichi, T.

AU - Umeda, S.

AU - Okuyama, H.

AU - Miyagawa, S.

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N2 - 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.

AB - 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.

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