Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens
Perota, Andrea (Laboratory of Reproductive Technologies)
Lagutina, Irina (Laboratory of Reproductive Technologies)
Duchi, Roberto (Laboratory of Reproductive Technologies)
Zanfrini, Elisa (Laboratory of Reproductive Technologies)
Lazzari, Giovanna (Fondazione Avantea)
Judor, Jean Paul (CHU Nantes)
Conchon, Sophie (CHU Nantes)
Bach, Jean-Marie (University of Nantes)
Bottio, Tomaso (Padova University School of Medicine)
Gerosa, Gino (Padova University School of Medicine)
Costa Farré, Cristina (Hospitalet de Llobregat)
Galiñanes, Manuel (Hospital Universitari Vall d'Hebron)
Roussel, Jean Christian (Nantes University Hospital (França))
Padler-Karavani, Vered (Tel Aviv University)
Cozzi, Emanuele (University Hospital of Padova (Pàdua, Itàlia))
Soulillou, Jean Paul (CHU Nantes)
Galli, Cesare (Fondazione Avantea)
Universitat Autònoma de Barcelona

Fecha:	2019
Resumen:	Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N -glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Materia:	Bioprosthetic Heart Valve (BHV) ; Cattle ; CMAH ; GGTA1 ; Knockout ; Neu5Gc ; Xenotransplantation ; αGal
Publicado en:	Xenotransplantation, Vol. 26 (may 2019) , ISSN 1399-3089

DOI: 10.1111/xen.12524
PMID: 31115108

13 p, 4.6 MB

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