RESUMEN
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
RESUMEN
Background: A unique feature of embryo metabolism is production of reactive oxygen species [ROS]. It is well established that during in vitro culture, ROS levels increase over normal ranges observed for embryos developed in vivo. This study evaluates and compares the stepwise pattern of ROS production during in vitro development of reconstructed goat embryos produced by zona-free method of somatic cell nuclear transfer [SCNT]. Furthermore, the pattern of ROS production of SCNT embryos were compared with zona free embryos derived from in vitro fertilization [IVF]
Materials and Methods: In this experimental study, zona-free oocytes, SCNT and IVF embryos at different stages of in vitro development [2, 4, 8, 16-cells, morula, and blastocyst] were used for assessment of ROS production using 2, 7-dichloro dihydroflourescein diacetate [DCHFDA] probe and the result were presented as fold increase or decrease relative zona free oocytes
Results: The relative level of ROS compared to metaphase-II [MII] oocytes insignificantly decrease during early stages post embryo reconstitution and regained its value by 8-cell and morula stage and, significantly increase compared to MII oocytes by blastocyst stage
Conclusion: The pattern of ROS change in SCNT embryos is similar to zona free IVF derived embryos, except it decrease from two cell stage and regain its value at morula stage. The sudden rise in ROS at blastocyst stage, further emphasizes the special need of IVF and SCNT derived embryos during this stage of development
RESUMEN
Objective: This research intends to unravel the temporal expression profiles of genes involved in three developmentally important signaling pathways [transforming growth factor-beta [TGF-beta], fibroblast growth factor [FGF] and wingless/int [WNT]] during pre- and peri-implantation goat embryo development
Materials and Methods: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the downstream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 [D7] blastocysts and day-14 [D14] blastocysts] were separately prepared in triplicate for comparative real-time reverse transcriptase polymerase chain reaction [RT-PCR] using the selected gene sets
Results: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-beta, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-beta, FGF and WNT components with renewal thereafter
Conclusion: The results suggested that TGF-beta, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of pre- and peri-implantation goat embryo development
RESUMEN
Background: Little is understood about the regulation of gene expression during early goat embryo development. This study investigated the expression profile of 19 genes, known to be critical for early embryo development in mouse and human, at five different stages of goat in vitro embryo development [oocyte, 8-16 cell, morula, day-7 blastocyst, and day 14 blastocyst]
Materials and Methods: In this experimental study, stage-specific profiling using real time-quantitative polymerase chain reaction [RT-qPCR] revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on their respective mRNA profiles
Results: The gradual pattern of reduction in the maternally stored transcripts without renewal thereafter [cluster-1: Lifr1, Bmpr1, Alk4, Id3, Ctnnb, Akt, Oct4, Rex1, Erk1, Smad1 and 5] implies that their protein products are essential during early cleavages when the goat embryo is silent and reliant to the maternal legacy of mRNA. The potential importance of transcription augment at day-3 [cluster-2: Fzd, c-Myc, Cdc25a, Sox2] or day-14 [cluster-3: Fgfr4, Nanog] suggests that they are nascent embryonic mRNAs which intimately involved in the overriding of MET or regulation of blastocyst formation, respectively. The observation of two expression peaks at both day-3 and day-14 [cluster-4: Gata4, Cdx2] would imply their potential importance during these two critical stages of pre-and peri-implantation development
Conclusion: Evolutionary comparison revealed that the selected subset of genes has been rewired in goat and human/goat similarity is greater than the mouse/goat or bovine/goat similarities. The developed profiles provide a resource for comprehensive understanding of goat preimplantation development and pluripotent stem cell engineering as well
RESUMEN
Several predisposing and risk factors are introduced as main causes of coronary atherosclerosis which is the main cause of myocardial infarction [MI]. In recent years, chronic and persistent infections are considered as such factors. This study is basically on determination of seropositivity to Chlamydia pneumonia to reveal the role of acute and chronic infections due to these bacteria as a risk factor for MI. 273 serum samples from MI patients and 109 samples from control group were examined by using commercial quantitative ELISA kits to measure specific anti Chlamydia pneumonia antibodies [IgM and IgG]. Two groups were matched for age and sex. IgM titers [ELISA] were negative in all patients and control cases, indicating lack of acute Chlamydial infection, but IgG titers were positive in 273 patients [94.4%], [mean average titer: 108 RU/ml] and in 109 control cases [84.4%] [mean average of titer: 61.9 RU/ml]. We found no significant relationship between seropositivity to Chlamydia pneumonia antibodies [lgG] with MI [P=0. 104]. In this study, no significant relationship was observed between serpositivity to Chlamydia pneumonia and subsequent incidence of MI. It seems that a large scale serological study with a diagnostic molecular methods for detection of genome in biopsy tissue of atherosclerotic coronary artery will be more helpful to reveal the expected relationship