40 TREATMENT OF DONOR CELLS AND ITS EFFECT ON INTERSPECIES NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 138
Author(s):  
M. A. Hashem ◽  
D. P. Bhandari ◽  
S. K. Kang ◽  
B. C. Lee
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Development Rate ◽  
Serum Starvation ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Significant Difference ◽  
Morula Stage

The present study was undertaken to examine the effect of donor cells, under a variety of treatment effects, on the development of goral porcine reconstructed embryos. Three experiments were performed, each with a one-way completely randomized design involving 3 to 4 replicates of all. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved (SS), and fully confluent stages of goral cells when reconstructed with porcine enucleated oocytes. In Experiment II, the effects of 2 antioxidants, β-mercaptoethanol (β-ME, 10 �M) and cysteine (2 mM), were examined after cells were fully confluent without serum starvation for 4 h. In Experiment III, the effect of different levels of dimethylsulfoxide (DMSO) at 0%, 0.5%, and 1.0% were tested, after 4 h of treatment, on the development rate after reconstruction with enucleated porcine oocytes. From the results, it appears that there were no significant (P > 0.05) differences from cleavage to morula among cyclic, SS, and fully confluent stages of the cell cycle. None of the treated group reached the blastocyst stage. There were no significant differences at the fused, 2- to 4-cell, and morula stages of embryo development after treatment of the donor cells with β-ME and cysteine before nuclear transfer. However, in the case of 8- to 16-cell stages, there were significant differences between β-ME and cysteine; the donor cells treated with β-ME had a better development rate than those treated with cysteine. No significant differences were observed in fusion, 2- to 4-cell, 8- to 16-cell, blastocyst, and hatching blastocyst stages at the 0.0, 0.5, and 1.0% levels of DMSO. However, there were statistically significant (P < 0.05) differences observed at the morula stage of embryo development. When donor cells were treated for 4 h with 0.5 and 1.0% levels of DMSO, goral-porcine reconstructed embryos reached the morula stage. From the results it can be concluded that goral somatic cells can be de-differentiated in porcine oocytes after treated with antioxidants and DMSO.

scholarly journals Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

2017 ◽  
Vol 7 (7) ◽  
pp. 2065-2080 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Justin C St. John
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Trichostatin A ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Genetic Integrity ◽  
Rna Seq ◽  
Mtdna Copy Number ◽  
Donor Cells

Abstract The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

scholarly journals Identifying Biomarkers of Autophagy and Apoptosis in Transfected Nuclear Donor Cells and Transgenic Cloned Pig Embryos

2019 ◽  
Vol 19 (1) ◽  
pp. 127-146
Author(s):  
Ju-Young Lee ◽  
Sang Hwan Kim ◽  
Jong Taek Yoon
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Transfected Cells ◽  
Autophagy Inhibitor ◽  
Dermal Cell ◽  
Donor Cells ◽  
Significant Difference ◽  
Dermal Cells

AbstractIn this study, we first investigated the effects of 3-methyladenine (3-MA), an autophagy inhibitor, and the inducer – rapamycin (RAPA) on the incidence of programmed cell death (PCD) symptoms during in vitro development of porcine somatic cell nuclear transfer (SCNT)-derived embryos. The expression of autophagy inhibitor mTOR protein was decreased in porcine SCNT blastocysts treated with 3MA. The abundance of the autophagy marker LC3 increased in blastocysts following RAPA treatment. Exposure of porcine SCNT-derived embryos to 3-MA suppressed their developmental abilities to reach the blastocyst stage. No significant difference in the expression pattern of PCD-related proteins was found between non-transfected dermal cell and transfected dermal cell groups. Additionally, the pattern of PCD in SCNT-derived blastocysts generated using SC and TSC was not significantly different, and in terms of porcine SCNT-derived embryo development rates and total blastocyst cell numbers, there was no significant difference between non-transfected cells and transfected cells. In conclusion, regulation of autophagy affected the development of porcine SCNT embryos. Regardless of the type of nuclear donor cells (transfected or non-transfected dermal cells) used for SCNT, there was no difference in the developmental potential and quantitative profiles of autophagy/apoptosis biomarkers between porcine transgenic and non-transgenic cloned embryos. These results led us to conclude that PCD is important for controlling porcine SCNT-derived embryo development, and that transfected dermal cells can be utilized as a source of nuclear donors for the production of transgenic cloned progeny in pigs.

80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY

2006 ◽  
Vol 18 (2) ◽  
pp. 148
Author(s):  
J. F. Yang ◽  
S. H. Yang ◽  
Y. Y. Niu ◽  
Q. Zhou ◽  
W. Z. Ji
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Methylation Pattern ◽  
Serum Starvation ◽  
Global Dna Methylation ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Morula Stage ◽  
Asymmetric Methylation

Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.

scholarly journals Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽  
2001 ◽  
pp. 801-808 ◽  
Author(s):  
JL Liu ◽  
MK Wang ◽  
QY Sun ◽  
XR Zhang ◽  
LK Jiang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Donor Cell ◽  
Serum Starvation ◽  
Fresh Tissue ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 147-156 ◽  
Author(s):  
Hong-Bo Liu ◽  
Pei-Ru Lv ◽  
Xiao-Gan Yang ◽  
Xiao-E Qin ◽  
Dao-Yuan Pi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Mammalian Species ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Contact Inhibition ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
New Born ◽  
Reconstructed Embryos

SummaryMiniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglet's testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10–14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10μg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).

39 DEVELOPMENTAL COMPETENCE OF RECONSTRUCTED EMBRYOS PRODUCED BY NUCLEAR TRANSFER BETWEEN CYNOMOLGUS MONKEY FIBROBLAST CELLS AND RABBIT OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 128 ◽  
Author(s):  
Y. Hosoi ◽  
T. Yamochi ◽  
N. Kawata ◽  
M. Takenoshita ◽  
S. Ohta ◽  
...  
Keyword(s):  
Cynomolgus Monkey ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Genetic Material ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Fibroblast Cells ◽  
Cell Stage ◽  
Reconstructed Embryos ◽  
Donor Cells

Interspecies nuclear transfer has been used as an invaluable tool for studying nucleus-cytoplasm interactions and it may also be used for rescuing endangered species whose oocytes are difficult to obtain. In this study, we investigated interaction of the cynomolgus monkey cell as a nuclear donor with the rabbit oocyte as a host cytoplasm. Whole cynomolgus fibroblast cells were injected into the rabbit enucleated oocytes (cynomolgus-rabbit cloned embryos) and cultured in TCM-199 and RPMI 1640 culture media. Rabbit-rabbit cloned embryos we used as control in this study. Karyotype analyses confirmed that genetic material of blastocysts was derived from the cynomolgus donor cells at blastocyst stage. Mitochondrial constitution analysis of the cynomolgus-rabbit cloned embryos indicated that mitochondria from both donor cells and enucleated oocytes coexisted. After culture for 168 h post-nuclear transfer, all cynomolgus-rabbit cloned embryos in TCM-199 were arrested at the 8-cell stage, but some of them developed to the blastocyst stage in RPMI 1640 (11/59, 18.6%). In this experiment, the nutrition requirement in vitro and the cleavage rate at each 24 h were examined. When TCM-199 was supplemented with lactate, some of these embryos developed to the blastocyst stage (15.3%, 2/13). This means that cynomolgus-rabbit cloned embryos might be controlled by the donor nucleus even in these early developmental stages. However, the timing of cleavage of cynomolgus-rabbit cloned embryos is very similar to that of the rabbit-rabbit cloned embryos. Time of cleavage may depend on the protein accumulated in the cytoplasm. In the prolonged culture of reconstructed embryos on feeder cells, adhesion cells were observed. These cells are also very similar to the cells derived from cynomolgus embryos by the same method. Our results suggest that: (1) a cynomolgus nucleus can co-ordinate with rabbit oocyte cytoplasm in early embryo development, (2) the 8- to 16-cell stage block in the cynomolgus-rabbit cloned embryos may due to the same reason as that in the cynomolgus embryos, and (3) ooplasmic factors that control time of cleavage are highly conservative between the species.

Effects of donor sex and different cell treatments on development of yak–bovine interspecies nuclear transfer embryos

2008 ◽  
Vol 5 (1) ◽  
pp. 55-60
Author(s):  
Liu Ying ◽  
Zhu Shi-En ◽  
Li Rong ◽  
Wang Li-Li ◽  
Wang Hai-Ping ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Cell Group ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Significant Difference ◽  
Interspecies Nuclear Transfer

AbstractThe purpose of this study was to evaluate the effects of donor sex, treatments of cell cycle synchronization and donor nuclei obtained from fresh or frozen–thawed conditions on developmental competence of yak–bovine interspecies nuclear transfer embryos. Bovine (Bos taurus) oocytes were used as recipients and yak (Bos grunniens) ear fibroblast cells were used as donors. Results indicated that the development rate of male blastocysts was higher than that of female (56.6% versus 39.5%, P<0.05), whereas cleavage and total cell number showed no difference between the two groups. No significant difference was observed in the development and quality of blastocysts with donor cells treated by serum starvation or contact inhibition, and there was no significant difference in embryo development with fresh or frozen–thawed donor cells, whereas the cleavage rate in the group of frozen–thawed cells was significantly lower than that of the fresh cell group (54.5% versus 78.2%, P<0.05). The results demonstrated that donor sex could impact the developmental competence of yak–bovine interspecies nuclear transfer embryos, whereas different treatments of cell cycle synchronization and freezing had little influence.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

35 IN VITRO BOVINE EMBRYO DEVELOPMENT AFTER NUCLEAR TRANSFER BY HANDMADE CLONING USING A MODIFIED WOW CULTURE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 126 ◽  
Author(s):  
C. Feltrin ◽  
F. Forell ◽  
L. dos Santos ◽  
J. L. Rodrigues
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Inner Diameter ◽  
Group 2 ◽  
Group 1 ◽  
Handmade Cloning

The effect of the microenvironment on embryo development during in vitro culture of zona-free embryos after nuclear transfer is still unclear. The aim of this experiment was to determine the effect of the dimensions of the well (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) culture system on the in vitro development of handmade cloned bovine embryos to the blastocyst stage. Appropriately ground steel needles were pressed slightly by hand to the bottom of the well of a polystyrene four-well dish (176740, Nunc, Life Technologies AS, Roskilde, Denmark). Embryos were produced by the handmade cloning (HMC) technique (Vajta et al. 2003 Biol. Reprod. 68, 571-578) with modifications, using primary cultures of skin fibroblast cells from an adult cow as nuclear donors. Cumulus-oocyte complexes were in vitro-matured in M-199 supplemented with 10% estrous cow serum (ECS), FSH, hCG, and estradiol (E2) for 17 h. After maturation, cumulus cells were removed by pipetting. Following zona pellucida removal in 0.5% protease (Sigma, Brazil), zona-free oocytes were incubated for 15 min in 5 mg/mL cytochalasin B (Sigma) and subsequently hand-bisected and screened for nuclear material under UV light after incubation in 10 mg/mL bisbenzimide (Hoechst 33342). Next, two enucleated halves and one donor cell were aggregated after a quick exposure to phytohemagglutinin (PHA) and subsequently fused by two electrical DC pulses of 1 kV/cm for 20 �s, in a BTX 453 chamber coupled to an ECM 2001 Electro Cell Manipulator System (BTX, Inc., San Diego, CA, USA), with additional exposure to brief pre- and post-fusion AC pulses of 15 V. Reconstructed embryos were chemically activated in 5 mM ionomycin (Sigma) for 5 min, followed by 2 mM 6-DMAP (Sigma) for 2.5 h. Finally, activated reconstructed cloned embryos were in vitro-cultured in one of two WOW culture systems (larger vs. smaller micro-wells) in 4-well plates containing 400 mL modified SOF medium supplemented with 10% ECS, under mineral oil, at 5% CO2, 5% O2 and 90% N2, and 39�C for 7 days. In Group 1 (large-size micro-well), embryos were cultured in individual cylindrical micro-wells with an inner diameter and depth of approximately 280 and 250 mm, respectively, whereas in Group 2 (small size micro-well), embryos were cultured in individual conical micro-wells with approximately 130 mm inner diameter and 150 mm depth. Data analysis was performed by the chi-square test. After four replicates, cleavage rates were significantly higher (P < 0.05) in Group 2 (51/63, 80.9%) than in Group 1 (43/67, 64.1%). Embryo development to the blastocyst stage was also greater (P < 0.05) in the small micro-wells (16/63, 25.3%) than in the large ones (8/67, 11.9%). In summary, these results show a significant increase in cleavage and blastocyst developmental rates in handmade cloned embryos cultured in a modified WOW system using individual small size micro-wells, suggesting that a small, tighter micro-well provides favorable in vitro conditions for embryo development.

27 SODIUM CHLORIDE TREATMENT OF CELL MEMBRANE-PERMEABILIZED NUCLEAR DONOR CELLS FACILITATES THE DISPLACEMENT OF THE SOMATIC HISTONE H1 AND HMG-17 PROTEINS IN RECONSTRUCTED PORCINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 122
Author(s):  
L. Che ◽  
V. Bordignon
Keyword(s):  
Sodium Chloride ◽  
Cell Membrane ◽  
Chromatin Structure ◽  
Nuclear Transfer ◽  
Chromatin Binding ◽  
Salt Extraction ◽  
Reconstructed Embryos ◽  
High Order Chromatin Structure ◽  
Donor Cells ◽  
High Order Chromatin

Developmental efficiency of somatic cell-reconstructed embryos depends on extensive remodeling of chromatin structural components. Due to their importance for maintaining the high-order chromatin structure and controlling DNA functions, including replication, transcription, repair, and recombination, histones and other chromatin-binding proteins represent leading choice markers to investigate nuclear remodeling in reconstructed embryos. The main objective of this study was to investigate whether or not the exposure of cell membrane permeabilized nuclear donor cells to sodium chloride (salt-extraction) would facilitate the displacement of chromatin-binding proteins in reconstructed porcine embryos. Both linker histone H1 (H1) and high-mobility group (HMG) proteins are known to affect gene expression through the modulation of the high-order chromatin structure. Standard methods of oocyte enucleation and electrofusion were applied for embryo reconstruction using in vitro-matured oocytes and control or salt-extracted fetal fibroblast cells. For salt-extraction, confluent cell cultures were washed with Ca2+/Mg2+-free Hank's balanced salt solution (HBSS); cells were permeabilized by incubation with 1 µg/mL of streptolysin O at 37°C for 30 min in HBSS, and then maintained in Tris-NaCl buffer (10 mM Tris-HCl, 0.5 mM MgCl2, 0.7 M NaCl, 1 M sucrose) for 5 min. After salt-extraction, cells were rinsed and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM CaCl2 for 1 h at 37.5°C for membrane resealing prior to nuclear transfer. Reconstructed embryos were activated using ionomycin (15 µM/5 min) and strontium chloride (Sr2+; 10 mM/4 h), and then cultured in PZM-3 medium. Immunostaining for H1 and HMG-17 was performed in nuclear donor cells and embryos at different stages after reconstruction. The time required for H1 displacement in transplanted nuclei was reduced by the salt-extraction treatment (Table 1). Salt-extracted cells showed a stronger HMG-17 cytoplasmic signal compared to control cells. The proportion of HMG-17-positive reconstructed embryos at 1, 3, and 6 h was 54 vs. 19, 57 vs. 44, and 75 vs. 62, for control and salt-extracted cells, respectively. These data suggest that salt-extraction prior to nuclear transplantation enhances the remodeling of chromatin structure in embryos reconstructed with somatic cell nuclei. Table 1. Proportion (n) of H1-positive stained embryos after different times from parthenogenetic activation (PA) and nuclear transfer using control (NT-control) or salt-extracted (NT-extracted) cells This work was supported by a NSERC Discovery Grant to VB.

Sign in / Sign up

Export Citation Format

Share Document