55 CONSTRUCTION AND IN VITRO DEVELOPMENT OF FOLLISTATIN TRANSGENIC PORCINE EMBRYO BY SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 133
Author(s):  
Y. S. Mu ◽  
D. D. Jiang ◽  
Y. J. Huan ◽  
J. Zhu ◽  
H. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Cell ◽  
Pcr Analysis ◽  
Cleavage Rate ◽  
Cmv Promoter ◽  
Transfected Cells ◽  
Blastocyst Rate

Data from mice showed that follistatin can block myostatin activity in vivo and follistatin overexpressed in transgenic mice can induce dramatic increases in muscle mass. In order to determine whether this effect of follistatin have the same results in swine, we design experiment to make a follistatin transgenic porcine by somatic cell nuclear transfer. We hope to get a follistatin transgenic porcine that has double muscle mass. The present study aimed to construct follistatin transgenic embryos by somatic cell nuclear transfer. A follistatin (FST) expression vector, pFST-1, which contains a porcine FST cDNA (with modification of codon usage) driven by the human cytomegalovirus (CMV) promoter, was constructed. A SV40-neo expression cassette as a selection marker was inserted into pFST-1 to generate pST101, which was transfected into early-passage male primary porcine fetal fibroblast cells by the lipofection method; the transfected cells were selected with 400 mg mL–1 G418. The G418-resistant colonies were pooled. Cell genome DNA was extracted and PCR analysis showed that CMV promoter, follistatin CDS, and SV40 polyA can be detected, indicating that the expression construct had integrated in cell genome. RT-PCR analysis showed that pST101 cells contained higher amounts of FST cDNA compared with the non-transfected cells, indicating that the follistatin was overexpressed in the primary porcine cells. Then, pST101 cell as donor cell and non-transfected cells as control donor cell were used to construct somatic cell nuclear transfer (SCNT) embryos. All of these SCNT embryos were cultured in Porcine Zygote Medium-3. The cleavage rate and blastocyst rate was assessed on 48 h and Day 6. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). The cleavage rate of pST101 cell and control cell derived SCNT embryos were 83.6 ± 2.0% (267/318) and 84.0 ± 2.2% (139/167) respectively, there were no significant (P > 0.05) differences between them. The blastocyst rate from pST101 cell and control cell derived SCNT embryos were 21.4 ± 1.8% (65/318) and 15.0 ± 3.0% (26/167) respectively, the SCNT embryos derived from pST101 cell showed a higher (P < 0.05) blastocyst rate. These results indicated that follistatin transgenic porcine embryo can be successfully constructed and can develop in vitro to blastocyst with a higher rate than control SCNT embryo. Further study will be focused on the full development in vivo of the transgenic embryos and phenotype of transgenic piglets. This work was supported in part by the National High-tech Research and Development Program of China (2008AA101006) and Major Projects for Transgenic Animals of China (2008ZX08006-002).

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 25-33 ◽  
Author(s):  
N. Chen ◽  
S-L. Liow ◽  
R. Bin Abdullah ◽  
WK. Khadijah Wan Embong ◽  
W-Y. Yip ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Oocyte Retrieval ◽  
Development Rate ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Polarized Microscopy

SUMMARYSomatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 °C) without CO2 supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

61 EFFECT OF ROOM TEMPERATURE HOLDING PROCEDURE ON ABILITY OF OOCYTES TO MATURE AND DEVELOP IN VITRO AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 136 ◽  
Author(s):  
K. Song ◽  
J. Lee ◽  
J. Park ◽  
W. Lee ◽  
Y. Chun ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Room Temperature ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Maturation Rate ◽  
Vitro Development

In Korea, it takes time to transport the ovaries of mares to the laboratory because horses are slaughtered only on Jeju island. Also, initiation of in vitro maturation (IVM) may be a little more delayed because of the oocyte collection by scraping of the follicular wall. It was reported that holding procedure of equine oocytes before IVM did not affect the developmental competence after intracytoplasmic sperm injection (Choi et al. 2006 Theriogenology 66, 955–963). The aims of present study were 1) to investigate the meiotic competence of equine oocytes held before IVM according to the type of oocytes, and 2) to examine the in vitro development after somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes (COCs) were recovered by scraping and washing of the follicular wall with Dulbecco’s modified Eagle medium (D-MEM) supplemented with 0.05% PVA, and classified as compact (Cp) or expended (Ex) depending on the expansion of cumulus or granulosa cells. 2 types of IVM procedures were compared: 1) COCs were matured immediately in IVM medium (TCM-199 supplemented with 5 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS) at 38.5°C under 5% CO2 in air for 24 to 27 h, and then held in holding medium (40% TCM-199 with Earle’s salts, 40% TCM-199 with Hanks’ salts, and 20% FBS) at room temperature for 6 to 7 h (control); or 2) COCs were initially held in holding medium for 6 to 7 h, and then matured in IVM medium for 24 to 27 h (holding). For SCNT, matured oocytes (pooled) were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 2 mM 6-DMAP, and cultured in D-MEM supplemented with 10% FBS and 50 ng mL–1 EGF at 38.5°C under 5% CO2, 5% O2, and 90% N2 for 7 to 9 days. Cleavage and blastocyst formation were evaluated on Days 2 and 8, respectively. All analyses were performed using SAS (version 9.1; SAS Institute Inc., Cary, NC, USA). 4 replicates were conducted from May to June 2010. In Ex oocytes, the maturation rate of the holding group (71.4%; 10/16) was not different from that of the control (65.6%; 44/73), and the rate of degenerated oocytes (4.8%; 1/16) in the holding group was not different from that in the control (5.6%; 5/73). However, in Cp oocytes, the degeneration rate of the holding group (65.0%; 31/49) was higher (P < 0.001) than that of the control (28.4%; 23/83), and the maturation rate of the holding group (20.6%; 12/49) was slightly lower (P = 0.07) than that of the control (46.0%; 38/83). After SCNT, the cleavage rate of the holding group (66.7%; 8/9) was not different from that of the control (60.8%; 14/25), and the rates of blastocyst formation of the control and the holding group were 8.1% (2/25) and 16.7% (2/9), respectively. Although the holding procedure may influence to the degeneration of Cp oocytes, it is considered that the developmental competence of equine oocytes held before IVM is not affected after SCNT.

scholarly journals Meiotic arrest as an alternative to increase the production of bovine embryos by somatic cell nuclear transfer

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 32-40 ◽  
Author(s):  
F.M.C. Caixeta ◽  
R.V. Sousa ◽  
A.L. Guimarães ◽  
L.O. Leme ◽  
J.F.W. Sprícigo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Meiotic Arrest ◽  
Embryo Production

SummaryThis study aimed to evaluate the effect of meiotic arrest using phosphodiesterase type 3A (PDE 3A) inhibitors, cilostamide and C-type natriuretic peptide (NPPC), on pre-maturation (PM) of oocytes to be used in the production of cloned embryos. Nuclear maturation, in vitro embryo production (IVP), somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA), and total cells number of cloned embryos were evaluated. The results were analysed by chi-squared and Kruskal–Wallis test with a P-value <0.05 considered to be significant. Approximately 87.8% of the oocytes remained at germinal vesicle stage (GV) after 6 h of PM with 5 μM of cilostamide, confirming the meiotic block. Embryo development in IVP was similar (P > 0.05) between control and PM, both for cleavage (78.2% and 76.9%) and blastocyst (35.5% and 29.3%) rates. After SCNT, cleavage rate was also similar (P > 0.05) between control and PM (66% and 51.9%) however, blastocyst rate was lower (P < 0.05) in the PM group than in the control group (7.4% and 30.2%). After 6 h of PM with 100 nM of NPPC, approximately 84.9% of the oocytes remained at GV. No difference was found between control and PM in cleavage (69.2% and 76.1%) and blastocyst rates (37,4% and 35%) after IVP. Similarly, no differences between PM and control groups were observed for cleavage (69.2% and 68.4%) and blastocyst (24.4% and 21.5%) rates. SCNT and PA embryos from control or PM oocytes had similar total cell number. It can be concluded that PM for 6 h with 100 nM NPPC is feasible for cloned embryo production without affecting embryo outcome.

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 67-72 ◽  
Author(s):  
So Gun Hong ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
Min Jung Kim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Transfected Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

MicroRNA-145 Inhibitor Significantly Improves the Development of Bovine Somatic Cell Nuclear Transfer Embryos In Vitro

2016 ◽  
Vol 18 (4) ◽  
pp. 230-236 ◽  
Author(s):  
Wenzhe Li ◽  
Yongjie Xiong ◽  
Fengyu Wang ◽  
Xin Liu ◽  
Yang Gao ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

Sign in / Sign up

Export Citation Format

Share Document