Yoichi Takahagi

Remodeling of the Major Pig Xenoantigen by N-Acetylglucosaminyltransferase III in Transgenic Pig

We have been successful in generating several lines of transgenic mice and pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

Development of efficient strategies for the production of genetically modified pigs

Although pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs, the efficiency of production of transgenic offspring is generally plagued by 1% of the DNA-injected embryos. Therefore, a problem with this method is the need for large numbers of pronuclear stage embryos. One great advancement would be the use of in vitro-matured (IVM) oocytes for the purpose of transgenic pig production. High developmental competence of IVM oocytes was proven by transfer of parthenogenetic IVM oocytes. A combined method of sperm vectors with the IVM of oocytes would make the production of transgenic pigs remarkably feasible. Rate of blastocyst formation following intracytoplasmic sperm injection (ICSI) by frozen sperm was over 20%, and transgene was expressed in approximately 50% of blastocysts generated. Somatic cell nuclear transfer would enable more efficient and sophisticated genetic modification of the pig. Simultaneous comparison between two nuclear transfer methods by electro-fusion and intracytoplasmic injection revealed clear differences in the pattern of nuclear remodeling and development of the reconstructed embryos. To specify the donor cell type that allows efficient genetic modification and easy reprogramming or to establish such cell lines is a critical issue in pig cloning. We tested pre-adipocytes from the subcutaneous adipose tissue of adult pigs for nuclear transfer. Cell cycle synchronization by differentiation induction is unique to the pre-adipocytes. Frequency of apoptosis was low in the cells synchronized by differentiation induction compared with other synchronization methods, including serum starvation, confluency, and chemical treatment. It would be of great worth if cryopreserved clone embryos were available. We have demonstrated that cryopreservation of in vitro-produced porcine embryos as well as clone blastocysts is possible by our unique method.

Survival of adult islet grafts from transgenic pigs with N-acetylglucosaminyltransferase-III (GnT-III) in cynomolgus monkeys.

Abstract:  Background:  Because of a severe shortage of human donor pancreases, pig islets are considered to be an attractive donor source. Our previous in vitro study revealed that adult pig islets have strong non‐Galα1‐3Galβ1‐4GlcNAc‐R (α‐Gal) antigenicity, including the Hanganutziu‐Deicher (H‐D) antigen, especially in N‐linked sugars. In this study, the issue of whether islets from N‐acetylglucosaminyltransferase‐III (GnT‐III) transgenic pigs can prolong their survival in cynomolgus monkeys was examined.

Production of alpha 1,3‐Galactosyltransferase gene‐deficient pigs by somatic cell nuclear transfer: A novel selection method for gal alpha 1,3‐Gal antigen‐deficient cells

The objective of the present study was to isolate alpha 1,3‐galactosyltransferase (GalGT)‐gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT‐DKO cells, GalGT‐gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT‐null cells were separated using a biotin‐labeled IB4 lectin attached to streptavidin‐coated magnetic beads. After 15–17 days of additional cultivation, seven GalGT‐DKO cell colonies were obtained from a total of 2.5 × 107 GalGT‐SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that αGal antigens were not present in the cells of the cloned DKO pigs. Mol. Reprod. Dev. 75: 1372–1378, 2008.

Comparison of Electro-Fusion and Intracytoplasmic Nuclear Injection Methods in Pig Cloning

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.

Multiple marker effects of single nucleotide polymorphisms in three genes, AKIRIN2, EDG1 and RPL27A, for marbling development in Japanese Black cattle

Marbling in beef, measured by Beef Marbling Standard (BMS) number, is an economically important trait for beef cattle breeding and markets in Japan. We previously detected three single nucleotide polymorphisms (SNPs) associated with BMS number of Japanese Black in Oita prefecture: c.*188G>A in AKIRIN2, g.1471620G>T in EDG1 and g.3109537C>T in RPL27A. Here, we carried out single and multiple marker association analyses for the three SNPs in a different commercial Japanese Black population of 892 genotyped animals. The single marker analyses with the model including a single SNP showed significant associations of all SNPs with BMS number. The multiple marker analysis with the model including the main effects of the three SNPs and their interactions detected only significant main effects of g.1471620G>T and g.3109537C>T and a significant interaction between c.*188G>A and g.1471620G>T. These findings suggest the presence of inter-allelic interactions among genes affecting the development of beef marbling. For effective marker-assisted selection for BMS number, interactions among these markers need to be considered.