Swine physiology and anatomy share many features with those of human. Stable pig stem cell lines are not available and somatic cell nuclear transfer (SCNT) is the best way to obtain transgenic litter using genetically modified somatic cells. The knock-out of α1,3-galactosyltransferase gene (GAL-/-) overcame the hyperacute rejection in swine-non human primates xenografts but to prolong the lifespan of transplanted organs, the acute vascular rejection has to be prevented inserting human genes to inhibit complement (CD55), coagulation and inflammation (CD39, EPCR and TM) cascades. The aim of our study was to obtain tetra-transgenic GAL KO cloned pigs to use in solid organ xenotranplantation programs. The activity of pCAGGS promoter and utility of 5´MAR of chicken lysozyme were successfully tested creating live cloned green fluorescent pigs. After that different expression vectors were obtained for selected human genes and a reliable screening platform was set up using PK15 transgenic colonies. GAL-/- fibroblasts were separately cotransfected with CD55Hygro+CD39 and EPCRPuro+TM expression vectors and resistant clones were efficiently selected using Western blot (WB) and Immunocitochemistry (ICC). Two live piglets (DAFne and Aretusa) were obtained using one GAL-/-/CD55+ colony and CD55 expression was widely characterized by WB, ICC, Immunohystochemistry and FACS in all analysed tissues and organs. One GAL-/-/CD55+CD39+ stillborn piglet (090210) was achieved but expression of CD55 and CD39 was maintained only in muscles. Instead, no pregnancies were obtained using EPCR+TM+ colonies. These results suggested us that strong expression of: CD55 is compatible with life of cloned piglets and it has cytoprotective effects on endothelial cells; CD39 has lethal anticoagulant effects; EPCR and TM are lethal for cloned embryos aborted after 45 days of pregnancy. Tissue-specific or inducible promoters will be used in order to obtain better cloning efficiency.

COSTRUZIONE DI VETTORI DI ESPRESSIONE GENICA PER LA REALIZZAZIONE DI SUINI TRANSGENICI DESTINATI AGLI XENOTRAPIANTI / A. Perota ; Tutor: Alberto Maria Luciano ; Correlatore: Cesare Galli; Coordinatore: Fulvio Gandolfi. - : . Universita' degli Studi di Milano, 2010 Dec 14. ((23. ciclo, Anno Accademico 2010.

COSTRUZIONE DI VETTORI DI ESPRESSIONE GENICA PER LA REALIZZAZIONE DI SUINI TRANSGENICI DESTINATI AGLI XENOTRAPIANTI.

A. Perota
2010

Abstract

Swine physiology and anatomy share many features with those of human. Stable pig stem cell lines are not available and somatic cell nuclear transfer (SCNT) is the best way to obtain transgenic litter using genetically modified somatic cells. The knock-out of α1,3-galactosyltransferase gene (GAL-/-) overcame the hyperacute rejection in swine-non human primates xenografts but to prolong the lifespan of transplanted organs, the acute vascular rejection has to be prevented inserting human genes to inhibit complement (CD55), coagulation and inflammation (CD39, EPCR and TM) cascades. The aim of our study was to obtain tetra-transgenic GAL KO cloned pigs to use in solid organ xenotranplantation programs. The activity of pCAGGS promoter and utility of 5´MAR of chicken lysozyme were successfully tested creating live cloned green fluorescent pigs. After that different expression vectors were obtained for selected human genes and a reliable screening platform was set up using PK15 transgenic colonies. GAL-/- fibroblasts were separately cotransfected with CD55Hygro+CD39 and EPCRPuro+TM expression vectors and resistant clones were efficiently selected using Western blot (WB) and Immunocitochemistry (ICC). Two live piglets (DAFne and Aretusa) were obtained using one GAL-/-/CD55+ colony and CD55 expression was widely characterized by WB, ICC, Immunohystochemistry and FACS in all analysed tissues and organs. One GAL-/-/CD55+CD39+ stillborn piglet (090210) was achieved but expression of CD55 and CD39 was maintained only in muscles. Instead, no pregnancies were obtained using EPCR+TM+ colonies. These results suggested us that strong expression of: CD55 is compatible with life of cloned piglets and it has cytoprotective effects on endothelial cells; CD39 has lethal anticoagulant effects; EPCR and TM are lethal for cloned embryos aborted after 45 days of pregnancy. Tissue-specific or inducible promoters will be used in order to obtain better cloning efficiency.
LUCIANO, ALBERTO MARIA
GANDOLFI, FULVIO
Xenotrapianto ; transgenesi ; CD55 ; CD39 ; EPCR ; TM ; suino ; clonazione ; effetto citoprotettivo endoteliale
Settore VET/01 - Anatomia degli Animali Domestici
COSTRUZIONE DI VETTORI DI ESPRESSIONE GENICA PER LA REALIZZAZIONE DI SUINI TRANSGENICI DESTINATI AGLI XENOTRAPIANTI / A. Perota ; Tutor: Alberto Maria Luciano ; Correlatore: Cesare Galli; Coordinatore: Fulvio Gandolfi. - : . Universita' degli Studi di Milano, 2010 Dec 14. ((23. ciclo, Anno Accademico 2010.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/150110
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