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Accomplishment of canine cloning through in vitro matured oocytes: a pioneering milestone
Kuk Bin Ji,Kangsun Park,Dongern Kim,Eunyoung Kim,Tae Young Kil,김민규 한국축산학회 2024 한국축산학회지 Vol.66 No.3
The in vitro maturation (IVM) rate of canine oocytes remains low compared to other mammals due to their unique reproductive characteristics. This study aimed to explore the effect of hormone supplementation during the IVM of canine immature oocytes on nuclear maturation and subsequently assess its potential application in canine somatic cell nuclear transfer (SCNT). Immature oocytes were collected and cultured in an IVM medium supplemented with hormones (follicle-stimulating hormone [FSH] and progesterone [P4]) or without hormones (control) for 24 hours. The maturation rates of oocytes in the hormone-treated group (94.92 ± 3.15%) were significantly higher than those in the control group (61.01 ± 4.23%). Both in vitro and in vivo matured oocytes underwent NT to evaluate their utility, and the fusion rates were higher in the in vitro matured group than those in the vivo matured group, not significant between in vivo and in vitro matured group (73.28% and 82.35%, respectively). As a result, 14 fused embryos from the in vitro matured group were transferred into two surrogates, with one surrogate achieving a successful pregnancy and delivering four puppies. Whereas in the in vivo matured group, 85 fused embryos were transferred to 8 surrogate mothers, leading to three surrogates becoming pregnant and delivering one, four, and two puppies. The pregnancy rates were not significant between both groups (50% and 37.50%), but the number of offspring exhibited a significant difference (28.57% and 8.23%). In conclusion, we achieved a remarkable milestone by successfully producing cloned puppies using in vitro matured oocytes, underscoring the feasibility of canine cloning from in vitro recovered oocytes. It is important to note that this study focused only on immature oocytes after ovulation and only during the estrus stage. Further research targeting other stages of the estrous cycle could potentially enhance canine cloning efficiency.
Application of the modified handmade cloning technique to pigs
( Eun Ji Lee ),( Kuk Bin Ji ),( Ji Hye Lee ),( Hyun Ju Oh ),( Tae Young Kil ),( Min Kyu Kim ) 한국축산학회(구 한국동물자원과학회) 2021 한국축산학회지 Vol.63 No.2
Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.
Eun Ji Lee,Kuk Bin Ji,Kang Sun Park,Kyeong Yeob Kim,Beom Sik Kim,Kyu hyun Kim,Ryeong Eun Kim,Eun Young Kim,Ji Hye Lee,Ju Lan Chun,Min Kyu Kim 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06
Somatic cell nuclear transfer(SCNT) using micromanipulator have been used as a traditional cloning technique and applied various researches during the last decades. However the micromanipulator is expensive and relatively longer training period is required to operate it efficiently. Handmade cloning(HMC) is an alternative cloning method in a simplified way compared to the traditional cloning. Here, we suggested modified porcine handmade cloning(mHMC) as a new approach to clone porcine embryos as a substitute of the traditional SCNT. In mHMC, a nucleus was removed by an aspiration method by using a glass pipette, instead of bisection method. In this study, we investigated the efficiency of the enucleation method in mHMC by assessing the developmental competence of embryos in comparison with the traditional SCNT. The efficiency of enucleation was evaluated based on the rate of the accuracy and oocyte survivability. The accuracy of enucleation was lower in mHMC compared to those in SCNT(98.01±0.57 vs. 83.83±2.47), and the rate of survived oocytes was also lower in mHMC(96.50±0.84 vs. 90.10±2.11, respectively). And the developmental competence was assessed. The blastocyst rate was significantly higher in mHMC group(13.53±2.08 vs. 20.48±0.99). The levels of apoptosis and ROS were investigated to evaluate embryo quality. The expression of ROS and apoptosis-related genes showed no difference between groups. And the relative expressions of mRNA of pluripotency genes and reprogramming genes were evaluated. Although DNMT1 and DNMT3α were not differently expressed in two groups, the expression of the one of pluripotent gene, Oct4 was significantly higher in mHMC. In conclusion, based on the comparable results of mMHC and SCNT, the mHMC could be a suitable alternative technique to clone embryos in cost effective way compared to traditional SCNT.
김현국(Hyun Kuk Kim),윤지열(Ji Yeol Yoon),이은영(Eun Young Lee),이창근(Chang Keun Lee),박중열(Joong Yeol Park),유빈(Bin Yoo),문희범(Hee Bom Moon) 대한내과학회 2002 대한내과학회지 Vol.63 No.4
Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder that has defects in glucose-6-phosphatase (G6Pase) in liver, kidney and intestinal mucosa. The defect leads to inadequate conversion of glucose-6-phospate to glucose in the liver and thus makes affected individuals susceptible to fasting hypoglycemia, hyperuricemia, lactic acidemia and hyperlipidemia. Hyperuricemia has been observed in a considerable number of patients and in some of those, clinical gout has occurred. Inhibited tubular secretion of uric acid due to hyperlacticacidemia and ketonemia, and overproduction of uric acid have been postulated as a mechanism for hyperuricemia in patients with GSD-Ia. A 30-year-old male was admitted with fatigue, foot pain and multiple gouty tophi on knee, ankle, and elbow. GSD-Ia and gout were confirmed by analysis of the G6Pase gene and tophi aspiration respectively. He was treated with allopurinol and uncooked cornstarch. After treatment, foot pain improved and the number and size of tophi were decreased.(Korean J Med 63:421-425, 2002)
Neuronal over-expression of human Alzheimer's disease related genes in canines
Chanuka Kulatunga,Dong Eon Kim,JI Hye Lee,Kuk Bin Ji,Eun Ji Lee,Kyeong Yeob Kim,Beom Sik Kim,Kyu Hyun Kim,Ryeong Eun Kim,Yoon Seok Nam,Min Kyu Kim 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
The early-onset familial Alzheimer's disease (EOFAD/ FAD), the less common type of Alzheimer's disease (AD) currently affects a vast number of individuals worldwide. This type is being inherited as an autosomal dominant fashion. Missense mutations on Amyloid precursor protein (APP) and Presenilins 1 and 2 (PSEN1 & PSEN2) are known as major genetic factors in FAD. Conversely, missense mutations on microtubule-associated protein tau (MAPT) are also thought to involve. Up to date, several triple-transgenic animal models with muted forms of the human APP, PSENs and MAPT have been reported. Compared to other animals, canines are more emotional and their disease signs can be easily diagnosed. This attempt was to develop a triple transgenic canine model for the AD. We have obtained the coding sequences of APP, PSEN1 and MAPT from Dana-Farber/Harvard Cancer Center DNA resource core at HMS and incorporated several common AD mutations. The transgenic construct is composed of hNSE (ENO2) promoter-driven three AD genes fused together with modified 2A sequences. It was transfected into the canine fetal fibroblasts which were then used to perform somatic cell nuclear transfer (SCNT). The viable transgenic embryos were obtained after in vitro culture and the GFP was detected. In this study, we have successfully produced viable triple transgenic canine cloned embryos using SCNT technique. These transgenic canine embryos will be further developed into canines with FAD. The transgenic canines will be a good candidate in the AD research field.
Regulatory Roles of Oocyte Secreted Factors in Canine Cumulus-oocyte Complexes and Granulosa Cells
Lili Zhuang,Dong Eon Kim,Chi Sun Yun,Kuk bin Ji,Eun Ji Lee,Gyeong Yeob Kim,Ji Hye Lee,Min Kyu Kim 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05
Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.