Dataset Information

Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs.

ABSTRACT: After the knock-out (KO) of ?1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.

SUBMITTER: Kwon DN

PROVIDER: S-EPMC4070623 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs.

Kwon Deug-Nam DN Lee Kiho K Kang Man-Jong MJ Choi Yun-Jung YJ Park Chankyu C Whyte Jeffrey J JJ Brown Alana N AN Kim Jae-Hwan JH Samuel Melissa M Mao Jiude J Park Kwang-Wook KW Murphy Clifton N CN Prather Randall S RS Kim Jin-Hoi JH

Scientific reports 20130101

After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those o ...[more]

PMID: 23760311

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Project description:BackgroundAnti-Galα1,3Galβ-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb₃) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods.MethodsThe expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb₃ synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples.ResultsAortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb₃ on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb₃ was also totally absent, whereas a fucosylated form of iGb₃ was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals.ConclusionsThese results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses.

Project description:Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn't been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.

Dataset Information

Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs.

Publications

Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs.

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