Treatment of Donor Cell/Embryo with Different Approaches to Improve Development After Nuclear Transfer

2014 ◽  
pp. 101-111 ◽  
Author(s):  
Eiji Mizutani ◽  
Sayaka Wakayama ◽  
Teruhiko Wakayama
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Embryo

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.

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
K. Inoue ◽  
N. Ogonuki ◽  
H. Miki ◽  
S. Noda ◽  
S. Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
High Rate ◽  
Full Potential ◽  
Cell Nuclei ◽  
Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

90 GERM-LINE TRANSMISSION OF DONOR MITOCHONDRIAL DNA IN NUCLEAR TRANSFER-DERIVED COWS

2007 ◽  
Vol 19 (1) ◽  
pp. 162
Author(s):  
K. Takeda ◽  
K. Kaneyama ◽  
M. Tasai ◽  
S. Akagi ◽  
M. Yonai ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Transfer ◽  
Germ Line ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Single Strand Conformation Polymorphism ◽  
Loop Region ◽  
D Loop ◽  
Germ Line Transmission ◽  
Host Embryo

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Distribution of donor mtDNA found in offspring of NT-derived founders may also vary from donor–host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to their progeny. NT cows were originally produced by fusion of enucleated oocytes with Jersey (J) or Holstein (H1) oviduct epithelial cells, or Holstein (H2) or Japanese Black (B) cumulus cells, as previously reported (Goto et al. 1999 Anim. Sci. J. 70, 243–245; Yonai et al. 2005 J. Dairy Sci. 88, 4097–4110; Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Transmission of donor cell mtDNA was analyzed by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis of the mitochondrial D-loop region. Eleven NT founder cows were analyzed, 4 (2 = J-NT, and 2 = H1-NT) of them were heteroplasmic whereas 7 (1 = J-NT, 1 = H1-NT, 2 = H2-NT, and 3 = B-NT) were homoplasmic for the host embryo mitochondria. The proportions of donor mtDNA detected in one J-NT cow was 7.7%, and those of other cow lineages were &lt;2%. Heteroplasmic NT cows delivered a total of 9 progeny. Four of the 9 progeny exhibited heteroplasmy with high percentages of donor cell mtDNA populations (52%, 37%, 17%, and 43%). The other 5 progeny were obtained from heteroplasmic NT cows, and all samples of the 10 progeny obtained from the homoplasmic NT cows did not harbor detectable donor cell mtDNA. A genetic bottleneck in the female germ-line will generally favor the transmission of a single mitochondrial population, leading to a return to homoplasmy. Thus, some of progeny maintained heteroplasmy with a higher ratio than that of their NT mothers, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrated that donor mtDNA in NT cows could be transmitted to progeny with varying efficiencies, in a lineage-specific fashion.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

38 RABBIT NUCLEAR TRANSFER AND IN VIVO-FERTILIZED EMBRYOS FAIL TO EXPRESS A MOUSE Oct-4 PROMOTER-DRIVEN EGFP REPORTER GENE

2007 ◽  
Vol 19 (1) ◽  
pp. 137 ◽  
Author(s):  
R. Hao ◽  
A. Wuensch ◽  
R. Klose ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Dna Methylation ◽  
Fluorescence Microscopy ◽  
Reporter Gene ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Bovine Embryos ◽  
Egfp Gene ◽  
Gene Construct ◽  
Fetal Fibroblasts

Reprogramming of a donor cell genome during somatic cell nuclear transfer (SCNT) is largely dependent on appropriate expression of 'pluripotency'? genes, such as Oct-4 (POU5F1). Recently, we transfected bovine fetal fibroblasts with GOF18-ΔPE-EGFP, a reporter gene construct for the Oct-4 promoter and assessed the expression of Oct-4 after SCNT (Wuensch et al. 2006 Reprod. Fertil. Dev. 18, 144). Our previous study on DNA methylation reprogramming revealed that rabbit in vivo-fertilized and cloned embryos differ from bovine embryos in respect to this epigenetic modification (Shi et al. 2004 Biol. Reprod. 71, 340–347), suggesting differences in the mechanism of epigenetic reprogramming between these two species. In this study, we tested whether GOF18-ΔPE-EGFP could be used to monitor Oct-4 expression in rabbit cloned embryos. The reporter gene construct included the EGFP gene flanked by a 9-kb fragment of the murine Oct-4 upstream region with a deletion in the proximal enhancer (PE) and a 9-kb fragment containing the nontranscribed murine structural Oct-4 gene. The 21.2-kb fragment GOF18-DPE-EGFP was released from the vector backbone by NotI digestion and purified with QIAquickGel Extraction Kit (Qiagen, Hilden, Germany) after gel electrophoresis. Four stable transfected colonies of rabbit fetal fibroblasts (RFF), none of which exhibited green fluorescence, were used for SCNT. The resulting embryos were examined on Days 2–5 by fluorescence microscopy. To detect endogenous Oct-4 expression, in vivo-fertilized embryos were stained with anti-mouse Oct-3 antibody and then incubated with secondary Alexa 488-conjugated goat anti-mouse antibody. The most prominent endogenous Oct-4 expression was detected in in vivo-fertilized embryos at the morula and blastocyst stages. Depending on the donor cell line used for nuclear transfer, cleavage and blastocyst rates ranged from 56 to 97% and from 33 to 49%, respectively. When a total of 230 cloned embryos at the 2-to 16-cell stages and 93 cloned morulae and blastocycts were examined by fluorescence microscopy, none of the examined embryos exhibited fluorescence signals indicating the lack of Oct-4 promoter activity. Taking into account the fact that both cloned and in vivo-fertilized rabbit embryos have specific patterns of DNA methylation reprogramming, which are different from that of bovine embryos, we injected GOF18-ΔPE-EGFP gene constructs into pronuclei of in vivo-fertilized zygotes. None of the 74 injected embryos, which were examined at the 2-cell to blastocyst stages, showed fluorescence signals. Our results demonstrate that rabbit nuclear transfer and in vivo-fertilized embryos are unable to activate a mouse Oct-4 promoter-reporter construct. Potential reasons include incompatibilities between mouse Oct-4 promoter sequences and rabbit transcription factors as well as specific mechanisms of epigenetic reprogramming in the rabbit. This work was supported by the Bayerische Forschungsstiftung.

Sign in / Sign up

Export Citation Format

Share Document