scholarly journals 70PRODUCTION OF CLONES BY FIBROBLAST NUCLEAR TRANSFER FROM AN X-AUTOSOME TRANSLOCATION CARRIER COW

2004 ◽  
Vol 16 (2) ◽  
pp. 156
Author(s):  
G.J. Rho ◽  
R. Kasimanickam ◽  
W.H. Johnson ◽  
E. Semple ◽  
D.H. Betts ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Primary Cultures ◽  
Donor Cell ◽  
Low Oxygen ◽  
Singleton Pregnancy ◽  
Internal Organs ◽  
Translocation Carrier ◽  
Cell Complexes ◽  
Autosome Translocation ◽  
Skin Biopsies

Poor reproductive outcome associated with chromosome anomalies is well documented in humans and domestic animals. In cattle, female carriers of Robertsonian and X-autosome translocations tend to be repeat breeders, probably due to synaptic difficulties during meiotic prophase of gametogenesis. Although viable offspring have been obtained through somatic cell nuclear transfer (NT) using adult or fetal cells from normal animals in various species, there have been no reports to date on the application of this technology to translocation carriers. Since NT circumvents meiotic problems encountered by translocation carriers, we used this approach to generate cloned embryos, fetuses and calves from a subfertile Limousin-Jersey crossbred cow previously identified as a carrier of an X-autosome translocation. Primary cultures were established from ear skin biopsies, and used at the 5th or 6th passage for NT. Recipient oocytes were enucleated at 19h post-maturation (hpm), fused with individual fibroblasts by a single electrical pulse (1.4KVcm−1, 40μs) and activated at 24hpm with a combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1). Reconstructed eggs were cultured in SOF at 39°C in a humidified low oxygen atmosphere. In 33 runs involving 2470 oocyte-donor cell complexes, cleavage and blastocyst rates were 88% (2173/2470) and 36% (889/2470), respectively. Two or three blastocysts (Day 7±1) were transferred into each recipient, previously synchronized with a combination of CIDR, GnRH and PGF2α. Ultrasonography was performed at Days 28 to 60 and at Days 90 and 150. Pregnancy was confirmed on Day 28 in 10 of a total of 22 recipients, 2 of which were later found to be carrying twin fetuses. Of 60 embryos transferred, 11 (18.3% of embryos) survived to Day 42, 6 (10%) to Day 60, and 4 (6.6%) to Day 90. A Day-94 fetus was surgically retrieved to examine the synaptic pattern of meiocytes in fetal ovaries. The fetus and internal organs were normal in appearance, and of normal size (16.5cm C-R length). The X-autosome translocation was confirmed in blood cultures, and synaptic anomalies involving chromosome 23 and the X chromosome were detected in fetal ovaries. Another clone was delivered by C-section at 276 days but died within 1h of delivery, while one singleton pregnancy is still ongoing at >200 days. These results demonstrate that NT can be used to produce embryos, fetuses and offspring from an X-autosome translocation carrier, with the potential to facilitate study of synaptic behaviour of female germ cells, and X-inactivation in different cell lineages of cloned blastocysts, and to generate individuals with otherwise poor reproductive prospects. [Supported by NSERC, Canada and OMAFRA]

47 BLASTOCYST DEVELOPMENT RATE OF CLONED BOVINE EMBRYOS USING SERIAL NUCLEAR TRANSFER OF CELLS CONTAINING AN X-AUTOSOME-TRANSLOCATED CHROMOSOME t(Xp+;23q-)

2006 ◽  
Vol 18 (2) ◽  
pp. 132
Author(s):  
W. A. King ◽  
B.-G. Jeon ◽  
D. H. Betts
Keyword(s):  
Nuclear Transfer ◽  
Primary Cultures ◽  
Calf Serum ◽  
Development Rate ◽  
Blastocyst Development ◽  
Cell Numbers ◽  
2Nd Generation ◽  
Autosome Translocation ◽  
Chi Square Analysis ◽  
Serial Nuclear Transfer

Somatic cell nuclear transfer (SCNT) has been utilized to study various genetic and epigenetic contributions of specific biomedical diseases and developmental events by using various donor cell types such as mature lymphocytes, brain tumor cells, and other unique genotypes. Previously, we produced cloned fetuses and offspring derived from SCNT of adult ear skin fibroblasts obtained from a sub-fertile cow harboring an X-autosome translocation as a model to study X-inactivation and chromosome dynamics during female meiosis. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned calf with the X-autosome translocation t(Xp+;23q-) compared to the original adult fibroblast donor containing the same chromosome translocation. Primary cultures of cells were established in DMEM +15% fetal calf serum (FCS). To serve as nuclear donors, cells at 5-7 passages were cultured for 5 days until confluent. Oocytes matured for 18 h in TCM-199 with hormones were removed of their chromatin, and reconstructed by transfer of donor cells and fusion with two DC pulses (1.2 kV/cm, 15 �s), delivered by a BTX 2000 Electro Cell Minupulator (BTX, Inc., San Diego, CA, USA), in 0.28 M mannitol containing 0.01 mM MgCl2. After 1 h of fusion, the eggs were activated with 5.5 �M ionomycin for 5 min, followed by 11 �g/mL cyclohexamide for 5 h. The eggs were cultured for 9 days in L-SOF at 39�C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2. Chi-square analysis revealed no significant (P > 0.05) differences in the rates of cleavage, blastocyst frequencies, and cell numbers between the 1st and 2nd generation cloned embryos. Cleavage rates were 87.4% and 85.4% for 1st and 2nd generation cloned embryos, respectively. The frequencies of blastocyst development and hatched blastocyst formation on Day 9 were 41.4% (91/220) and 38.7% (92/238), and 26.4% (58/220) and 22.7% (54/238) for the 1st and 2nd generation cloned embryos, respectively. The numbers of total cells and inner cell mass (ICM) cells of Day 9 blastocysts were 183 and 52, respectively, in the 1st generation embryos and 167 and 51 cells in the 2nd-generation cloned embryos. In summary, 2nd generation cloned embryos derived from fibroblasts of a cloned calf with an X-autosome translocated chromosome showed embryo development and cell numbers similar to those of the 1st generation clones. These results demonstrate that serial nuclear transfer does not improve the blastocyst development rate of cloned embryos containing the X-autosome translocation t(Xp+;23q-). This work was funded by OCAG, OMAF, and CRC.

43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 140
Author(s):  
N. Hornen ◽  
W. A. Kues ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
P. Hassel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Primary Cultures ◽  
Donor Cell ◽  
Stem Cell Population ◽  
High Density Culture ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Normal Offspring

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 � 10.5% (mean � SD) vs. 83.7 � 9.2%; fetal fibroblasts: 64.8 � 17.3% vs. 82.5 � 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 � 3.5% vs. 24.9 � 8.6%). In the second experiment, FSSCs of 3 different sizes (<14 �m, 15–20 �m, >21 �m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 � 3.1 vs. 91.3 � 10.1 vs. 92.3 � 5.1; cleavage rates: 83.5 � 7.9 vs. 83.1 � 1.6 vs. 83.2 � 5.8; blastocyst rates: 15.3 � 7.9 vs. 17.6 � 6.8 vs. 10.4 � 2.7, respectively). In the third experiment, FSSCs 15–20 �m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3� 7.3% vs. 86.8 � 5.3%; cleavage rates: 86.1 � 6.7% vs. 80.7 � 5.9%; blastocyst rates: 21.4 � 5.6% vs. 18.4 � 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.

24 Evaluation of Latrunculin A for the Activation of Hand-Made Cloning (HMC) Porcine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 151
Author(s):  
F. K. Castañeda ◽  
N. G. Canel ◽  
G. V. Landschoot ◽  
A. De Stéfano ◽  
R. J. Bevacqua ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Uv Light ◽  
Electric Pulse ◽  
Polar Body ◽  
Primary Cultures ◽  
Donor Cell ◽  
Activation Process ◽  
Latrunculin A ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) is an important biotechnological tool. However, production rates of viable offspring remain low. One possible cause of this low efficiency is chromosomal losses during early activation process (Liu et al. 2015 Cell. Reprogram. 17, 463–471). The use of actin inhibitors that block second polar body extrusion during activation protocols might be a strategy to avoid such losses. The objective of this work was to compare the efficiency of the use of 2 actin inhibitors during the activation of hand-made cloning (HMC) porcine embryos. One of the compounds used was latrunculin A (LatA), which joins directly to actin monomers, preventing their assembly to the filaments. The other was cytochalasin B (CB), which is commonly used for activation protocols. It binds to the growing actin filaments and prevents their elongation. For this purpose, in vitro-matured cumulus–oocyte compexes were deprived of their cumulus and zonae pellucidae cells by mechanical and enzymatic treatments. Oocytes were randomly distributed in 2 experimental groups (HMC) and 2 parthenogenetic control groups (PA). For HMC groups, oocytes were bisected using a microblade and the resulting hemioocytes were stained with Hoechst 33342 and observed under UV light to identify those that had lost the metaphase II plate. Adult skin fibroblasts from primary cultures were used as nuclear donors. For nuclear transfer, 2 hemicytoplasts were fused to a donor cell by an electric pulse of 1.42 kV/cm for 30 μs. After 2 h of nuclear reprogramming, the reconstituted embryos were activated by an electric pulse of 1.2 kV/cm for 80 μs and incubated with cycloheximide (CHX, 10 μg mL−1 , 3 h) in combination with one of the actin inhibitors: LatA 2 μM (CHX-LatA goup) or CB 2.5 μg mL−1 (CHX-CB group). The PA groups were subjected to the same activation treatments (PA-CHX+LatA and PA-CHX+CB groups). All embryos were cultured in SOFaa medium, using an adaptation of the well-of-the-well (WOW) system (microwells), in a humidified atmosphere with 5% CO2 in air at 39°C. Cleavage, morulae, and blastocysts rates were evaluated at Days 2, 4, and 7-8, respectively. At least 3 replicates were performed per group. Results are presented in Table 1. Our results demonstrate that the production of embryos by HMC activated with CHX-LatA is as efficient as that with CHX-CB, the protocol currently used in SCNT protocols. Further research is needed to study its effect on chromosomal complements and long-term development. Table 1.Effect of activation with cycloheximide (CHX) and latrunculin A (LatA) on in vitro development of hand-made cloning (HMC) porcine embryos (% ± SD in parentheses)

scholarly journals Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. O. Olsson ◽  
A. H. Tinson ◽  
N. Al Shamsi ◽  
K. S. Kuhad ◽  
R. Singh ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Large Scale ◽  
Donor Cell ◽  
Blastocyst Formation ◽  
Short Term ◽  
Embryo Formation ◽  
Large Expansion ◽  
Breed Differences ◽  
Breed Comparison

AbstractCloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

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 Lessons Learned from Somatic Cell Nuclear Transfer

2020 ◽  
Vol 21 (7) ◽  
pp. 2314 ◽  
Author(s):  
Chantel Gouveia ◽  
Carin Huyser ◽  
Dieter Egli ◽  
Michael S. Pepper
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Lessons Learned ◽  
Epigenetic Reprogramming ◽  
Area Of Interest ◽  
Legal Implications ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

scholarly journals Synaptonemal complex analysis of an X-autosome translocation-carrier in the domestic pig

1991 ◽  
Vol 23 (Suppl 1) ◽  
pp. S222 ◽  
Author(s):  
DAF Villagomez ◽  
M Świtoński ◽  
B Singh ◽  
KRS Fisher ◽  
I Gustavsson ◽  
...  
Keyword(s):  
Synaptonemal Complex ◽  
Complex Analysis ◽  
Translocation Carrier ◽  
Domestic Pig ◽  
Autosome Translocation ◽  
Synaptonemal Complex Analysis
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