scholarly journals In vitrodevelopment of canine somatic cell nuclear transfer embryos in different culture media

2015 ◽  
Vol 16 (2) ◽  
pp. 233 ◽  
Author(s):  
Dong-Hoon Kim ◽  
Jin-Gu No ◽  
Mi-Kyung Choi ◽  
Dong-Hyeon Yeom ◽  
Dong-Kyo Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Media

33 PROPAGATION OF ELITE LIFESAVER DOGS BY SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 164
Author(s):  
B. C. Lee ◽  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Calcium Ionophore ◽  
Defined Medium ◽  
German Shepherd ◽  
Surrogate Mothers ◽  
Working Dogs

Canine somatic cell nuclear transfer (cSCNT) has been used as a useful tool for propagation of elite working dogs. In 2009, 7 cloned dogs were successfully produced using somatic cells derived from the excellent drug-sniffing dog of Korea Customs Service. All cloned dogs perfectly performed drug detection in Incheon International Airport. The objective of the present study was to compare the efficiency of the 2 activation culture media to clone the retired Baekdu, a veteran rescue dog that performed lifesaving activities worldwide for 6 years in Korea National Emergency Management Agency (NEMA). Ear tissue was collected from a 10-year-old male German Shepherd and fibroblasts were cultured for cSCNT. The cells were injected into the perivitelline space of enucleated in vivo-matured dog oocytes, fused with electric stimulation using an electro cell fusion apparatus (Nepa Gene Co. Ltd.), and activated chemically. In the activation protocol, 2 different types of media were tested to investigate the effect of proteins with undefined functions. The first medium was a modified synthetic oviduct fluid (mSOF), which is a complex culture medium with BSA that includes undefined functions. The second medium was the porcine zygote medium (PZM-5), which is a chemically defined medium with polyvinyl alcohol (PVA). The fused couplets were activated by mSOF medium supplemented with 1.9 nM DMAP (SOF-DMAP), and PZM-5 supplemented with 1.9 nM DMAP (PZM-DMAP) for 4 h, followed by 4 min of calcium ionophore treatment. Then, reconstructed oocytes were transferred into the uterine tube of naturally estrus-synchronized surrogate dogs. In the PZM-DMAP group, a total of 56 activated cloned embryos were transferred into 3 female recipient dogs, and a total of 64 activated cloned embryos from the SOF-DMAP group were transferred into 4 female recipients. Pregnancy diagnosis was performed using a SONOACE 9900 (Medison, Seoul, Korea) ultrasound scanner with 7.0-MHz linear-array probe between 30 and 35 days after embryo transfer. As a result, pregnancy was detected in 1 out of 3 surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and 1 pregnancy (25%) was detected in 4 surrogate mothers receiving cloned embryos from the SOF-DMAP group. Two pregnant dogs each gave birth to 1 healthy cloned puppy by cesarean section. This study shows that existence of proteins with undefined functions in activation medium did not affect the dog cloning. In addition, the number of elite working dogs in diverse fields can be increased by the NT technique using donor cells derived from small tissue of elite working dogs. This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, and TS Corporation.

293 EFFECT OF MEDIUM TYPE FOR CULTURE OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS ON PRE-IMPLANTATION DEVELOPMENT OF CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 294
Author(s):  
G. A. Kim ◽  
H. J. Oh ◽  
J. Kim ◽  
T. H. Lee ◽  
J. H. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Culture Media ◽  
Formation Rate ◽  
Donor Cells

Mesenchymal stem cells (MSC) have been known as useful donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that the culture condition of donor cells causes different results on preimplantation development of SCNT embryos. In this study, we investigated the patterns of gene expression of adipose-derived mesenchymal stem cells (ad-MSC) in different culture media (DMEM and RKME), and examined the effect of ad-MSC, with the gene expression changed, used as donor cells on the preimplantation development of cloned embryos. Canine ad-MSC were isolated from fat tissue of 3-year-old female beagle and were cultured in DMEM supplemented with 10% fetal bovine serum (MSC-DMEM) and RKME (MSC-MSC) provided from RNL Bio Corp. (Seoul, Korea). Total RNA was extracted from ad-MSC cultured in each culture medium. After synthesising cDNA of each sample, quantitative RT-PCR was done according to the Takara Bio Inc. guidelines and using the 7300 Real Time PCR Cycler System (Applied Biosystems, Carlsbad, CA, USA). The level of all tested gene transcription was normalized to β-actin expression levels. The relative quantification of gene expression was analysed by the 2–ΔΔCt method. The data from all experiments were analysed by Student’s t-test using a statistical analysis GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA). Significance was determined at P < 0.05. The stemness, the reprogramming-related gene expression level of donor cells of MSC-DMEM and MSC-MSC were compared. In order to confirm the effect of MSC cultured in 2 different culture media on somatic cell nuclear transfer, we performed interspecies somatic cell nuclear transfer (iSCNT). The enucleated bovine oocytes were injected, respectively, with donor cells of MSC-DMEM and MSC-MSC, and were fused by electrofusion. The iSCNT embryos were cultured in modified SOF at 38.5°C for 7 days in an atmosphere of 5% CO2 and 5% O2, and the developmental ability of iSCNT embryos was observed under the microscope. The MSC-MSC contained a significantly higher amount of Sox2, Nanog, Oct4, Stella, HDAC1, DNMT1, and MeCP2 than the MSC-DMEM, whereas the amount of Rex1 was not different in either MSC-MSC or MSC-DMEM. In the development ability of iSCNT embryos, MSC-DMEM embryos resulted in a 16-cell embryo formation rate that was higher than that of MSC-MSC embryos (9.09 and 5.30%, respectively; P < 0.05). However, the blastocyst formation rate was not different between MSC-DMEM embryos and MSC-MSC embryos (4.5 and 3.2%, respectively; P > 0.05). These results demonstrate that the gene expression of ad-MSC can be modified, by culture media, into a state where reprogramming is easily done. Even so, ad-MSC with gene expression changed by culture medium did not influence the developmental ability of blastocysts. In conclusion, the alteration of gene-related stemness and reprogramming in canine ad-MSC would not be able to effectively control reprogramming in SCNT. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311062-04-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.

scholarly journals A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Xiangpeng Dai ◽  
Jie Hao ◽  
Qi Zhou
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Culture Method ◽  
Early Embryo ◽  
Blastocyst Development

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and αMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10–30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

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