scholarly journals Studies on Gene Expression in Bovine Embryos Derived from Somatic Cell Nuclear Transfer

2009 ◽  
Vol 55 (1) ◽  
pp. 11-16 ◽  
Author(s):  
Ken SAWAI
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bovine Embryos

34 EVALUATION OF DIFFERENT SEQUENTIAL CULTURE SYSTEMS ON THE DEVELOPMENT AND QUALITY OF BOVINE EMBRYOS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 123
Author(s):  
R. F. Felmer ◽  
M. E. Arias ◽  
J. L. Riveros ◽  
G. A. Munoz ◽  
J. H. Rio
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Culture System ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Culture Systems

Different culture systems have been developed that support development of bovine embryos up to the blastocyst stage. However, the use of sequential culture systems has been studied less. The objective of the present study was therefore to examine the effect of 3 sequential culture systems, which involve the use of different culture media for early cleavage and later stage embryos, on the development and quality of bovine embryos generated by somatic cell nuclear transfer (SCNT). These systems were mainly based on different combinations of KSOM culture medium regularly used in our laboratory. Skin fibroblasts, at passage 5 to 6, were microsurgically placed into the perivitelline space evacuated during enucleation. Fusion was carried out by a single DC pulse of 1.7 kV cm–1 with an Electrocell Manipulator 830 (BTX Inc., San Diego, CA, USA) and activation by treatment of NT units in Ionomicin (5 μM, 4 min) and DMAP (1.9 mM) for 4 h. Embryo culture was carried out in 50-μL drops under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2, in a humidified atmosphere, according to the following sequential culture systems without co-culture: 1) KSOM + 0.4% BSA (FAF, A8806, Sigma, St. Louis, MO, USA) for 3 days and then KSOM + 5% FBS (characterized, Hyclone, Logan, UT, USA) to Day 7; 2) KSOM + 0.1% BSA for 3 days and then SOF + 0.8% BSA to Day 7 and 3) KSOM + 0.1% BSA for 3 days and then KSOM + 0.8% BSA to Day 7. Cleavage rate was evaluated on Day 3 and the number of blastocysts was recorded on Day 7. A total of 730 NT embryos randomly distributed were analysed for embryo development in 9 replicates and 5 blastocysts of each group were stained with bisbenzimide (Hoechst 33242) to assess the quality. ANOVA was used to test for statistically significant differences (P < 0.05) using Satgraphics Plus 2 Software. In cases where statistically significant differences were observed, a multiple comparison test was run using Tukey’s test. Sequential culture systems had no effect on cleavage rate (73, 76, and 73%, respectively). However, there was a significant difference (P < 0.01) in the rate of blastocysts. The sequential culture system consisting of KSOM + KSOM 5% FBS yielded a higher rate of blastocysts than other treatments (28, 18, and 16%, respectively). Despite this difference in embryo development, the quality of embryos as assessed by the total number of cells was not different (135 ± 6.5, 129 ± 7.5, and 128 ± 8.5, respectively). In conclusion, a sequential culture system consisting of KSOM + 0.4% BSA for 3 days and then KSOM + 5% FBS to Day 7 generated a higher number of cloned blastocyst than other treatments evaluated, although the quality of the embryos did not differ between treatments. Future studies are under way to establish the gene expression profile of NT embryos generated under these culture systems. The final aim is to evaluate the possibility of modulating the gene expression profile through changes in the culture medium composition. The provision of ovaries by our local slaughterhouse (Frigorifico Temuco) and funding support from FONDECYT 1080216 CONICYT, Chile, are gratefully acknowledged.

64 X-LINKED GENE EXPRESSION IN BOVINE (BOS TAURUS) MALE AND FEMALE IN VITRO- FERTILIZED AND SOMATIC CELL NUCLEAR TRANSFER-DERIVED BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 140
Author(s):  
M. Nino-Soto ◽  
G. Mastromonaco ◽  
P. Blondin ◽  
W. A. King
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Somatic Cell ◽  
Dosage Compensation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Regulatory Mechanisms ◽  
Linked Gene ◽  
Male And Female

Expression of some X-chromosome linked genes has recently been shown to be altered in bovine somatic cell nuclear transfer (SCNT) derived embryos (Wrenzycki et al. 2002 Biol. Reprod. 66, 127), implying that the regulatory mechanisms of X-linked transcription are affected by embryo in vitro production (IVP) methods. We analyzed the transcriptional pattern of X-linked genes (BIRC4, GAB3, HPRT1, MECP2, RPS4X, SLC25A6, and XIST) in bovine in vitro fertilized (IVF) and SCNT male and female blastocysts to determine X-inactivation status and changes resulting from IVP. We collected pools of male (n = 5 pools) and female (n = 3 pools) IVF-derived blastocysts (Bousquet et al. 1999 Theriogenology 51, 59) and male (n = 5 pools) and female (n = 3 pools) SCNT-derived blastocysts (Mastromonaco et al. 2004 Reprod. Domest. Anim. 39, 462). Each pool consisted of five blastocysts. Embryos were washed in phosphate buffered saline (PBS) + 0.1% polyvinyl alcohol (PVA), collected, and stored at -80�C. Total RNA was extracted with an Absolutely RNA Microprep kit (Stratagene, La Jolla, CA, USA), DNase I treated, and precipitated with isopropanol and linear acrylamide (Ambion, Inc., Austin, TX, USA) as a carrier. Reverse transcription was performed with Oligo-dT (Invitrogen, Burlington, Ontario, Canada) and Superscript II RT (Invitrogen). Transcript quantification was performed by quantitative real-time PCR using SYBR Green I (LightCycler system, Roche, Diagnostics, Laval, Quebec, Canada). Data analysis was performed with SAS (SAS Institute, Inc., Cary, SC, USA) using a mixed-model factorial ANOVA and with results presented as estimates of the median, ratios of estimates, and 95% confidence intervals with � = 0.05. IVF-derived male and female blastocysts possessed similar levels of the transcripts analyzed, suggesting successful dosage compensation at this developmental stage for embryos fertilized in vitro. XIST was not detected in male IVF embryos. GAB3 was not detected in any of the female groups and, in addition, HPRT1 transcripts were not detected in SCNT derived female embryos. Male and female SCNT-derived blastocysts possessed marked differences in their transcript levels, with males showing statistically significantly higher levels of BIRC4 and RPS4X and females possessing higher levels of MECP2 and SLC25A6 transcripts although differences between the latter two were not statistically significant. XIST was detected in both male and female SCNT blastocysts. We conclude that dosage compensation between male and female IVF blastocysts is achieved at this developmental stage for the transcripts examined. However, this pattern was markedly changed in the SCNT group, affecting especially female SCNT blastocysts, suggesting that the regulatory mechanisms of X-inactivation and X-linked gene expression are substantially altered in SCNT embryos probably due to aberrant epigenetic patterns and faulty genome reprogramming. We are currently analyzing X-linked transcription in male and female in vivo-derived blastocysts in order to compare this group with IVP-derived embryos. This work was funded by NSERC, CIHR, and CRC.

scholarly journals Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e108139 ◽  
Author(s):  
Maria Jesús Cánepa ◽  
Nicolás Matías Ortega ◽  
Melisa Carolina Monteleone ◽  
Nicolas Mucci ◽  
German Gustavo Kaiser ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Expression Profile ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Vitro Fertilization

28 TREATMENT WITH SUBEROYLANILIDE HYDOXAMIC ACID OR SODIUM BUTYRATE ON PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DERIVED FROM KIDNEY CELLS OF HUMAN HEME OXYGENASE-1 TRANSGENIC PIG

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
K.-Y. Song ◽  
J.-H. Moon ◽  
E.-J. Park ◽  
S.-J. Kim ◽  
Y.-B. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Sodium Butyrate ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Heme Oxygenase 1 ◽  
Kidney Cells ◽  
Cloning Efficiency ◽  
Blastocyst Formation

Because somatic cell nuclear transfer (SCNT) is influenced by many factors concerning a series of various steps, the cloning efficiency is low in so many species and it seems to be more serious in production of transgenic (TG) animals. Reprogramming of donor nucleus is one of the important factors that affects the developmental competence of SCNT embryos, and several epigenetic remodelling drugs have been used to improve the cloning efficiency. In this study, we examined the effect of suberoylanilide hydoxamic acid (SAHA) or sodium butyrate (NaBu) treatment on the development of porcine SCNT embryos derived from kidney cells of TG pig. Fully confluent porcine kidney cells expressing the human heme oxigenase-1 gene were used for nuclear donor. For SCNT, matured oocytes with 1st polar body were enucleated, electrically fused, and activated 1 h after fusion (Song et al. 2009 Mol. Reprod. Dev. 76, 611–619). Then, SCNT embryos were incubated in postactivation medium [PA; porcine zygote medium-5 (PZM-5) supplemented with 0.5% dimethyl sulfoxide] for 4 h (control), PA with 0.4 μg mL–1 demecolcine for 4 h (Dc), PA with 0.5 μM SAHA for 9 h (SAHA), or PA with 1 mM NaBu for 9 h (NaBu). After postactivation treatment, SCNT embryos were cultured in fresh PZM-5 for 7 days. The embryos were examined for cleavage and blastocyst formation on Days 2 and 7, respectively (the day of SCNT was designated Day 0). Total cell number of blastocysts was examined by counting the number of nuclei stained with Hoechst 33342 under ultraviolet light. Complementary DNA synthesised with total RNA extracted from blastocysts were used for qRT-PCR to determine HDAC2, HDAC6, and GAPDH gene expression. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism version 5.01 (Graphpad Software, San Diego, CA, USA). The cleavage rates (77.0–82.9%) of treated embryos were not different from that of control embryos (79.0%). Blastocyst formation was slightly increased in Dc- (36/132, 27.3%), SAHA- (34/125, 28.6%), and NaBu- (36/133, 27.3%) treated embryos than in control embryos (32/128; 25.0%), but the difference was not significant. Total cell numbers (45.2–47.5) of treated embryos were not different from that of control embryos (51.8). Expression of HDAC2 was higher in SAHA-treated embryos than in control and Dc-treated embryos (P < 0.05), but it was not different from that of NaBu-treated embryos. The relative expression of HDAC6 transcript was increased in SAHA- and NaBu-treated embryos, but there was no significant difference among all groups. Although SAHA or NaBu did not improve the pre-implantational development of porcine SCNT embryos derived from kidney cells of TG pig as assessed in this study, additional studies are needed to determine the effect of SAHA or NaBu on gene expression of pig TG embryos and developmental competency following embryo transfer according to the origin of donor cells. This study was supported by IPET (#311011-05-2-SB010), MOTIE (#10033839-2012-21) and the TS Corporation.

scholarly journals Microtubule distribution in somatic cell nuclear transfer bovine embryos following control of nuclear remodeling type

2010 ◽  
Vol 11 (2) ◽  
pp. 93 ◽  
Author(s):  
Dae-Jin Kwon ◽  
Yu-Mi Lee ◽  
In-Sun Hwang ◽  
Choon-Keun Park ◽  
Boo-Keun Yang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bovine Embryos ◽  
Nuclear Remodeling
Sign in / Sign up

Export Citation Format

Share Document