ABSTRACT
Objective: In the present study, we investigated the possible epigenotoxic effect of dimethyl sulfoxide [DMSO] on buffalo fibroblast cells and on reconstructed oocytes during buffalo-bovine interspecies somatic cell nuclear transfer [iSCNT] procedure and its effect on rate and quality of blastocyst which derived from these reconstructed oocytes
Materials and Methods: In this experimental study, cell viability of buffalo fibroblasts was assessed after exposure to various concentration [0.5, 1, 2 and 4%] of DMSO using MTS assay. The epigenetic effect of DMSO was also assessed in terms of DNA methylation in treated cells by flowcytometry. Reconstructed oocytes of buffalo-bovine iSCNT exposed for 16 hours after activation to non-toxic concentration of DMSO [0.5%] to investigate the respective level of 5-methylcytosine, cleavage and blastocyst rates and gene expression [pluripotent genes: OCT4, NANOG, SOX2, and trophectodermal genes: CDX2 and TEAD4] of produced blastocysts
Results: Supplementation of culture medium with 4% DMSO had substantial adverse effect on the cell viability after 24 hours. DMSO, at 2% concentration, affected cell viability after 48 hours and increased DNA methylation and mRNA expression of DNMT3A in fibroblast cells. Exposure of reconstructed oocytes to 0.5% DMSO for 16 hours post activation did not have significant effect on DNA methylation, nor on the developmental competency of reconstructed oocyte, however, it decreased the mRNA expression of NANOG in iSCNT blastocysts
Conclusion: Depending on the dose, DMSO might have epigenotoxic effect on buffalo fibroblast cells and reconstructed oocytes and perturb the mRNA expression of NANOG in iSCNT blastocysts
ABSTRACT
Objective: The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection
Materials and Methods: In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer [SCNT] and sperm mediated gene transfer [SMGT] approaches
Results: PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage [attP] sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction [PCR] and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique
Conclusion: The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein [EGFP] positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection [ICSI] method
ABSTRACT
Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region [DMR] in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies have shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception micro-environments often is occurred by assisted reproduction techniques [ART]. This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART
Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites [CpGs] located in this region
Result: The overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85 +/- 4.87% and 43.75 +/- 5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined [49.52 +/- 1.86% and 50%, respectively]
Conclusion: Considering the absence of in vivoproduced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks
Subject(s)
Animals, Laboratory , Female , Humans , Male , Blastocyst , Genomic Imprinting , In Vitro Techniques , CpG Islands , Epigenesis, Genetic , Reproductive Techniques, Assisted , IranABSTRACT
PhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene. Identification of a novel pseudo attP site. Genomic DNA was extracted from primary bovine fetal fibroblast cells, which were stably trans-fected with EGFP and phiC31 integrase cDNAs carrying vectors. An inverse PCR was carried out for production of mini-circle DNAs and followed by sequencing. A new specific pseudo attP site termed BF5 was identified in bovine genome. This site is located in an intergenic AT rich region on chromosome 5 with similar features of other mammalian attP pseudo sites. Furthermore, direct sequencing of generated attL site confirmed that site-specific transgene recombination was occurred at this site. This finding confirmed that phiC31 integrase could be feasible for production of transgenic animals for biotechnological applications
ABSTRACT
Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine [5-aza-dc; 0-0.3 microM], a DNA methyl transferase inhibitor, improved development and expression of Oct-4. In comparison with untreated cells, 0.01 and 0.08 microM 5-aza-dc treated cells insignificantly decreased the blastocyst rate [32.1% vs. 28.6% and 27.2%, respectively] while it was significant for 0.3 microM treated cells [6.5%]. Embryo quality as measured by the total cell number [TCN] decreased in a dose-related fashion, which was significant at 0.08 and 0.3 microM 5-aza-dc treated cells when compared with 0 and 0.01 microM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 microM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 microM 5-aza-dc remained unchanged. These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryo