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Production of Transgenic Male Piglet as Research Model for Alzheimer's Disease
Mi-Ryung Park,Kyong-Woon Kim,Jae-Seok Woo,Seongsoo Hwang,In-Sul Hwang1,Tae-Uk Kwak,Ji-Hyun Lim,Se-Pil Park 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10
Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the human AD related genes (APP, APPswedish, Presenilin 1, and Tau). Transgenic embryos were generated by SCNT of from ear fibroblasts expressing AD genes. A total of 1808 (average 258) SCNT embryos were transferred into 7 recipients. Pregnancy was successfully maintained in one recipient. We obtained 1 cloned male piglet from a surrogate gilts and the weight of piglet was 935 g. But, the male piglet died two days after birth. The piglet expressed AD related genes by PCR and western blot analysis. Transgenes were expressed in multiple tissues, and at especially high levels in brain. AD Tg pig might be very useful for studying the disease and for testing new therapeutics in preclinical studies of human AD.
Seongsoo Hwang,Keon Bong Oh,Dae-Jin Kwon,Sun A Ock,Jeong-Woong Lee,Jin-Ki Park,Gi-Sun Im 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s
Here we report the productions of genetically modified cloned Massachusetts General Hospital miniature pig (MGH minipig) using freshly thawed donor cells equilibrated with roscovitine. Fibroblasts were isolated from the ear skin of a 10-day-old male MGH minipig. The donor cells were divided into two groups: cultured for 3 days (culture) and freshly thawed with 500 nM roscovitine. The viability of the donor cells was significantly higher at 0 h (94.6±3.5) compared with 1 h (81.7±5.7) after thawing (p=0.028). After 1 hr of equilibration time, the proportion of G0/G1 stage in roscovitine group was not different from 0 hr group, but not in culture medium group (p<0.01), respectively. Although the developmental characteristics were not different in both methods, the pregnancy and delivery rate in freshly thawed group were significantly higher than that of culture group (p<0.01), respectively. In total, 12 TG cloned MGH minipigs were delivered and the individual cloning efficiency was from 0 to 2.54%. Taken together, the use of freshly thawed donor cells equilibrated with roscovitine may be helpful method to increase the productivity of the genetically modified cloned MGH minipigs.
Seongsoo Hwang,Keon Bong Oh,Tae‐Uk Kwak,Dae‐Jin Kwon,Dong‐Hoon Kim,Jae‐Seok Woo,Jin‐Ki Park,Gi‐Sun Im 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1
Pig parthenotes were able to develop in vivo for 30 days with normal morphology. In pig, during blastocyst elongation between day 10 and 12 of gestation, estrogen production and secretion by conceptus increases, serving not only as the signal for maternal recognition of pregnancy, but also as a stimulus for the production of proteins and growth factors within the uterine environment that initiate implantation. Cloning efficiency is still very low regardless of species. To increase the productive efficiency of (transgenic, TG) clones, an advanced somatic cell nuclear transfer (SCNT) method may need. Here we report the productions of transgenic cloned pigs using cloned embryos and parthenotes simultaneously. Fibroblasts were isolated from an ear skin of a 10‐day‐old NIH miniature pig. The ear fibroblast cells were transfected with the alpha1,3‐ Galactosyltransferase knock‐out/human CD46 knock‐in (GalT KO/hCD46 KI). For SCNT, the TG somatic cells were used as donor cells. Immediately after fusion confirmation, the TG cloned embryos and parthenotes were transferred into both oviducts of surrogates. The mean number of TG cloned embryos and parthenotes was 137 (±15.2) and 123(±27.1), respectively. The pregnancy and delivery rate was (55.6%, 10/ 18) (44.4%, 8/18), respectively. Totally 19 GalT KO/hCD46 KI cloned piglets were delivered. Among them, 11 piglets were survived and 8 piglets were born stillbirth. The healthy 5 piglets are still survived.
Characterization of α-Gal Epitope Expression in Skin derived from Transgenic Pigs
Seongsoo Hwang,In-Sul Hwang,Dae Jin Kwon,Tae-Uk Kwak,Hyun Yang,Mi-Ryung Park,Keon Bong Oh,Sun-A Ock,Gi-Sun Im,Jeong-Woong Lee 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10
The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. There are genes related to the expression of α-Gal epitope such as α1,3Galactosyltransferase gene (GT-/-) and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the expression of α-Gal epitope in the skin derived from GT-/- transgenic pig. The skin (7/1000 inches) was obtained by dermatome (Zimmer® Electric Dermatome) from one month old of wildtype (WT) and GT-/- piglets, respectively. The skins were fixed, dehydrated, cleaned, and embedded. To analyze the expression of α-Gal epitope, the paraffin section of WT and GT-/- were stained with BS-IB4 lectin and isoglobotrihexosylceramide synthase antibody. There was a strong BS-IB4 lectin signal in the skin of WT, but not detected in GT-/-. However, the iGb3s positive signals were stained in the skin of both WT and GT-/-. Taken together, it can be postulated that the knocked out of GT gene may not enough to inhibit the expression of α-Gal epitope. Further studies are needed to evaluate the functions of the double knock out of GT and iGb3s on the expression of α-Gal epitope.