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
Objective: For the first time, we used molecular signaling pathway enrichment analysis to determine possible involvement of miR-126 and IRS-1 in neurotrophin pathway
Materials and Methods: In this prospective study, validated and predicted targets [targetome] of miR-126 were collected following searching miRtarbase [http://mirtarbase.mbc.nctu.edu.tw/] and miRWalk 2.0 databases, respectively. Then, approximate expression of miR-126 targeting in Glioma tissue was examined using UniGene database [http://www.ncbi. nlm.nih.gov/unigene]. In silico molecular pathway enrichment analysis was carried out by DAVID 6.7 database [http://david. abcc.ncifcrf.gov/] to explore which signaling pathway is related to miR-126 targeting and how miR-126 attributes to glioma development
Results: MiR-126 exerts a variety of functions in cancer pathogenesis via suppression of expression of target gene including PI3K, KRAS, EGFL7, IRS-1 and VEGF. Our bioinformatic studies implementing DAVID database, showed the involvement of miR-126 target genes in several signaling pathways including cancer pathogenesis, neurotrophin functions, Glioma formation, insulin function, focal adhesion production, chemokine synthesis and secretion and regulation of the actin cytoskeleton
Conclusion: Taken together, we concluded that miR-126 enhances the formation of glioma cancer stem cell probably via down regulation of IRS-1 in neurotrophin signaling pathway
ABSTRACT
Background: Polycystic ovarian syndrome [PCOS] is a metabolic and endocrine disorder which is characterized by hyperandrogenism, anovulation or oligomenor-rhea and polycystic ovarian morphology. It is believed that modulation in metabo-lism of granulosa cells of PCOS patients may lead to infertility. One of the metabolic modulators is FNDC5 and its cleaved form, irisin. The axis of PGC1 Alpha - FNDC5 pathway is one of the main factors affecting cellular energy balance the purpose of this study was to evaluate this pathway in granulosa cells derived from PCOS mice model in comparison with control group
Methods: In the present study, PCOS mouse model was developed by injection of dehydroepiandrosterone [DHEA] hormone in 20 mice for a period of 20 days. Also, 20 uninjected mice were used as the control. Meanwhile, a vehicle group consisted of mice which received daily subcutaneous sesame oil injection [n=20]. Relative ex-pressions of PGC1á and FNDC5 in granulosa cells were evaluated by RT-qPCR. Analysis of gene expressions was calculated by the Delta Delta CT method and the relative levels of mRNA were normalized to GAPDH transcript levels. Differences in genes expression among three groups were assessed using one-way ANOVA, Tukey's Post Hoc test
Results: Our results showed that expression of FNDC5 was significantly reduced in granulosa cells of DHEA-induced PCOS mice compared with control and vehicle groups [p<0.05], while there was no significant differences in PGC1 Alpha expression among different groups
Conclusion: Down regulation of FNDC5 transcript level may contribute in metabol-ic disturbance of granulosa cells derived from PCOS ovary apart from PGC1 Alpha levels which remained unchanged
ABSTRACT
Objective: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector
Materials and Methods: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells
Results: In the present study, we developed a nonviral episomal vector named pLENSO/Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming
Conclusion: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future
Subject(s)
Induced Pluripotent Stem Cells , CpG Islands , Plasmids/genetics , Bacteria/geneticsABSTRACT
Objective: Liver X receptors [LXRs] are ligand-activated transcription factors of the nuclear hormonal receptor superfamily which modulate the expression of genes involved in cholesterol homeostasis. Hence, further unraveling of the molecular function of this gene may be helpful in preventing cardiovascular diseases
Materials and Methods: This experimental intervention study included twelve adult Wistar male rats [12-14 weeks old, 200-220 g] which were divided into the control [n=6] and training [n=6] groups. The training group received exercise on a motor-driven treadmill at 28 meters/minute [0% grade] for 60 minutes a day, 5 days a week for 4 weeks. Rats were sacrificed 24 hours after the last session of exercise. A portion of the liver was excised, immediately washed in ice-cold saline and frozen in liquid nitrogen for extraction of total RNA. Plasma was collected for high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], total cholesterol [TC] and triglycerides [TG] measurements. All variables were compared by independent t test
Results: A significant increase in LXR alpha transcript level was observed in trained rats [P<0.01]. Plasma HDL-C concentration was also significantly higher [P<0.01] in trained rats. There was a significant decrease in the concentrations of LDL-C [P<0.01] and TC [P<0.02], and the ratios of TC/HDL-C [P<0.001] and LDL/HDL-C [P<0.002] in trained rats. However, the TG concentration was unchanged [P>0.05]
Conclusion: We found that endurance training induces significant elevation in LXR alpha gene expression and plasma HDL-C concentration resulting in depletion of the cellular cholesterol. Therefore, it seems that a contributor to the positive effects of exercise in cardiovascular disease prevention is through the expression of LXR alpha, which is a key step in reverse cholesterol transport
Subject(s)
Animals, Laboratory , Male , Liver X Receptors , Lipids , Cholesterol, HDL , Liver , Rats, WistarABSTRACT
Objective: Due to recent progress in production of human embryonic stem cell-derived oligodendrocyte progenitor cells [hESC-OPCs] for ameliorating myelin disease such as multiple sclerosis [MS] and the role of purinergic signaling in OPCs development, we avaluated the profile of purinergic receptors expression during development of OPCs from hESC
Materials and Methods: In this experimental study, we used reverse transcription and quantitative polymerase chain reaction [RT-qPCR] to obtain more information about potential roles of purinergic receptors during in vitro production of hESC-OPCs. We first determined the expression level of different subtypes of purinergic receptors in hESCs, embryoid bodies [EBs], and hESC-OPCs. The effects of A1adenosine receptor [A1AR] activation on hESC-OPCs development were subsequently examined
Results: hESCs and OPCs had different mRNA expression levels of the AR subtypes. ARs mRNA were expressed in the EB stage, except for A2AAR. We observed expressions of several P2X [P2X1, 2, 3, 4, 5, 7] and P2Y [P2Y1, 2, 4, 6, 11-14] genes in hESCs. hESC-OPCs expressed different subtypes of P2X [P2X1, 2, 3,4,5,7] and P2Y [P2Y1, 2, 4, 6, 11-14]. Except for P2X1 and P2X6, all other P2X and P2Y purinergic receptor subtypes expressed in EBs. We also indicate that A1AR might be involved in modulating gene expression levels of cell cycle regulators in an agonist and/or dose-dependent manner
Conclusion: Elucidation of the expression pattern of purinergic receptors and the effects of different subtypes of these receptors in hESC-OPCs may have a promising role in future cell-based therapy or drug design for demyelinating disease
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Objective: Annexin A1 [ANXA1] is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species [ROS] production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium [MPP+]
Materials and Methods: In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 [IL-6], inducible nitric oxide synthase [iNOS] and nuclear factor-kappa B [NF-kappaB] were assessed by flow-cytometry, real-time quantitative polymerase chain reaction [RT-qPCR] and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells
Results: Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-kappaB protein in MPP+ treated PC12 cells
Conclusion: ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions
ABSTRACT
Objective: Peroxisome proliferator-activated receptor gamma [PPAR gamma] is a member of the PPAR nuclear receptor superfamily. Although PPAR gamma acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPAR gamma in order to address its role in cardiac cell differentiation of mouse embryonic stem cells [mESCs]
Materials and Methods: In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction [qPCR] was carried out to estimate level of gene expression. Furthermore, the requirement of PPAR gamma in cardiac differentiation of mESCs, during cardiac progenitor cells [CPCs] formation, was examined by applying the respective agonist and antagonist
Results: The obtained data revealed an elevation in the expression level of PPAR gamma during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPAR gamma inactivation via treatment with GW9662 [GW] reduced expression of CPC and cardiac markers
Conclusion: We conclude that PPAR gamma modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis
ABSTRACT
Background: Globozoospermia is a rare syndrome with an incidence of less than 0.1% among infertile men. Researchers have recently identified a large deletion, about 200 kbp, encompassing the whole length of DPY19L2 or mutations in SPATA16 and PICK1 genes associated with globozoospermia. The aim of this study was to analyze the DPY19L2 gene deletion using polymerase chain reaction technique for the exons 1, 48, 11 and 22 as well as break point [BP] [a] in globozoospermic men
Materials and Methods: In this experimental study, genome samples were collected from 27 men with globozoospermia [cases] and 36 fertile individuals [controls], and genomic analysis was carried out on each sample
Results: Deletion of DPY19L2 gene accounted for 74% of individuals with globozoospermia. DPY19L2 gene deletion was considered as the molecular pathogenic factor for the onset of globozoospermia in infertile men. By quantitative real-time polymerase chain reaction [qPCR], we genotyped DPY19L2 deletion and identified carriers within the population
Conclusion: This technique may be considered as a method for family counseling and has the potential to be used as a pre-implantation genetic diagnosis, especially in ethnic community with high rate of consanguineous marriages
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Objective: MicroRNAs [miRNA] are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis [MS]. Th17 and regulatory T [Treg] cells are two subsets of CD4[+] T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4[+] T-cell derived miR-223 during the relapsing-remitting [RR] phase of MS [RR-MS], as well as the expressions of Th17 and Treg cell markers
Materials and Methods: this experimental study used real-time quantitative polymerase chain reaction [qRT-PCR] to evaluate CD4[+] T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases [n=40] compared to healthy controls [n=12], along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 [FOXP3] and RAR-related orphan receptor ?t [ROR?t] in CD4[+] T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively
Results: miR-223 significantly upregulated in CD4[+] T-cells during the relapsing phase of RR-MS compared to the remitting phase [P=0.000] and healthy individuals [P=0.036]. Expression of ROR?t, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O [FOXO1] and FOXO3 were predicted by in silico studies
Conclusion: miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS
ABSTRACT
Objective: Nanotechnology focuses on materials having at least one dimension of less than 100 nanometers. Nanomaterials such as Nanosilver [NS] have unique physical and chemical properties such as size, shape, surface charge. NS particles are thought to induce neuronal degeneration and necrosis in the brain. It has been reported that NS particles generate free radicals and oxidative stress which alters gene expression and induces apoptosis. This study was designed to evaluate whether the detrimental effect of NS particles is through the activation of Procaspase-3 during fetal neural development
Materials and Methods: In this experimental study, thirty Wistar female rats at day one of pregnancy were semi-randomly distributed into three groups of ten. Group 1, the control group, had no treatment. From day 1 to the end of pregnancy, groups 2 and 3 received 1 and 10 ppm NS respectively via drinking water. Newborn rats were sacrificed immediately after birth and their brains were dissected and kept frozen. Total RNA, extracted from brain homogenates, was reverse transcribed to cDNA. Quantitative real-time polymerase chain reaction [PCR] analysis was undertaken to estimate the expression level of Procaspase-3
Results: Developmental exposure to NS induced neurotoxicity and apoptosis. This correlated with a significant increase in Procaspase-3 expression level especially at 10 ppm NS
Conclusion: The pro-apoptotic activity of NS in cells is likely to due to the dysregulation of Procaspase-3
ABSTRACT
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
Th17 cells are known to be involved in some types of inflammations and autoimmune disorders. RORC2 is the key transcription factor coordinating Th17 cell differentiation. Thus, blocking RORC2 may be useful in suppressing Th17-dependent inflammatory processes. The aim was to silence RORC2 by specific siRNAs in naive T cells differentiating to Th17. Time-dependent expression of RORC2 as well as IL-17 and IL-23R were considered before and after RORC2 silencing. In this experimental study, naive CD4+ T cells were isolated from human cord blood samples. Cytokines TGFbeta plus IL-6 and IL-23 were used to polarize the naive T cells to Th17 cells in X-VIVO 15 serum free medium. A mixture of three siRNAs specific for RORC2 was applied for blocking its expression. RORC2, IL-17 and IL-23R mRNA and protein levels were measured using qRT-PCR, ELISA and flow cytometry techniques. Pearson correlation and one-way ANOVA were used for statistical analyses. Significant correlations were obtained in time-dependent analysis of IL-17 and IL-23R expression in relation with RORC2 [R=0.87 and 0.89 respectively, p<0.05]. Silencing of RORC2 was accompanied with almost complete suppression of IL-17 [99.3%; p<0.05] and significant decrease in IL-23R gene expression [77.2%, p<0.05]. Our results showed that RORC2 is the main and the primary trigger for upregulation of IL-17 and IL-23R genes in human Th17 cell differentiation. Moreover, we show that day 3 could be considered as the key day in the Th17 differentiation process
Subject(s)
Humans , Cell Differentiation , Interleukin-17 , Receptors, Interleukin , RNA, Small Interfering , Nuclear Receptor Subfamily 1, Group F, Member 3ABSTRACT
In vitro simulation of developmental processes is an invaluable tool to shed light on the intrinsic mechanism of developmental biosystems such as central nervous system in mammals. Chick somites have been used to simulate the neural differentiation of human neural progenitor cells. In the present study, we aimed to indicate whether somites have the ability to express required neural differentiation factors at mRNA level. Chick embryos were isolated from the yolk surface of the fertilized eggs and somites were subsequently isolated from embryos under a dissecting microscope. Total RNA of the somites was extracted and RT-PCR carried out with specific primers of cerberus, chordin, FGF8, follistatin and noggin. Data showed that five aforementioned factors were co-expressed after 7 days in vitro by somites. We concluded that neural induction property of somites appeared by production of required neural differentiation factors including cerberus, chordin, FGF8, follistatin and noggin
ABSTRACT
The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 [DE3]. Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] of the purified phiC31 integrase confirmed the size of protein [70 kDa]. Finally, the functionality of purified phiC31 integrase was verified. The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration
Subject(s)
Integrases , DNA, Complementary , Cloning, Organism , Genetic Vectors , Gene Expression , DNA Nucleotidyltransferases , Electrophoresis, Polyacrylamide Gel , Polymerase Chain ReactionABSTRACT
The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter ystem is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein [EGFP] was controlled by the mouse Oct-4 promoter. In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs
Subject(s)
Animals, Laboratory , Green Fluorescent Proteins , Pluripotent Stem Cells , Embryonic Stem Cells , MiceABSTRACT
RNA interference-based gene silencing has recently been applied as an efficient tool for functional gene analysis. RORC2 is the key transcription factor orchestrating Th17 cells differentiation, the cells that are known as the pathogenic elements in various autoimmune diseases. The aim of this study was to design efficient siRNAs specific for RORC2 and to evaluate different criteria affecting their functionality. Three siRNA duplexes specific for RORC2 mRNA were designed. Th17 cells were produced from IL-6 and IL-1 treated cord blood CD4[+] T cells. The T cells were transfected with three different designed siRNAs against RORC2 and the expression of RORC2 gene was measured using quantitative real time PCR. Different levels of RORC2 down regulation were observed in the presence of each of the designed siRNAs. Efficient siRNA with 91.1% silencing activity met the majority of the established bioinformatics criteria while the one with 46.6% silencing activity had more deviations from these criteria. The more bioinformatics criteria are considered, the more functionality were observed for silencing RORC2. However, the importance of the type of criteria per se should not be neglected. Although all recommended criteria are important for designing siRNA but their value is not the same
Subject(s)
Humans , Gene Silencing , Th17 Cells , RNA, Small Interfering , RNA, MessengerABSTRACT
Peroxisome Proliferator Activated Receptor gamma [PPAR[gamma]], a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPAR[gamma] gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPAR[gamma]1 promoter region. Thus, expression pattern of PPAR[gamma]1 isoform is due to the potential transcription factors that could influence its promoter activity. PPAR[gamma], Retinoid X Receptor [RXR] and Vitamin D Receptor [VDR], as nuclear receptors could influence PPAR[gamma] gene expression pattern during several differentiation processes. During neural differentiation, PPAR[gamma]1 isoform expression reaches to maximal level at neural precursor cell formation. A vast computational analysis was carried out to reveal the PPAR[gamma]1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPAR[gamma]1 promoter was assessed in different cell lines. Results indicated that Rosiglitazone increased PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body [EB] formation. Furthermore vitamin D reduced PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPAR[gamma]1 isoform promoter. Also VDR/RXR heterodimers may decrease PPAR[gamma] expression through binding to its promoter
ABSTRACT
Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene. Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein coding sequence. In order to clone this region with a GC-content of 71.01%, a cocktail of ammonium sulfate buffer supplied with two additive components, betaine and dimethyl sulfoxide, and a high concentration of MgCl[2] was used. The modulated polymerase chain reaction composition significantly improved the amplification of GC-rich DNA target sequences. Improved amplification of this region was due to reduction in the formation of secondary structures by the GC-rich region. Therefore, this polymerase chain reaction composition could be generally used to facilitate the amplification of other GC-rich DNA sequences as verified by amplification of different GC rich regions
ABSTRACT
Background: Multiple births occur frequently in some Iranian sheep breeds, while infertility scarcely occurs. Mutation detection in major fecundity genes has been explored in most of Iranian sheep flocks over the last decade. However, previously reported single nucleotide polymorphisms [SNPs] for bone morphogenetic protein receptor-[BMPR]-1B and growth differentiation factor] GDF9[known to affect fertility have not been detected. This study was conducted to assess whether any significant mutations in GDF9 were extracted from slaughtered ewe ovaries of Iranian Afshari sheep breed
Materials and Methods: Ovaries defined as poor, fair, and excellent quality based on external visual appearance of follicles were used for histology and RNA extraction processes. High quality RNAs underwent reverse transcriptase-polymerase chain reaction [RT-PCR] from GDF9 mRNA, and the products sequenced
Results: No streak ovaries, which are considered indicators of infertility due to homozygocity for some mutations in GDF9 and BMP15, were found. Sequencing results from GDF9 cDNA showed that G2 [C471T], G3 [G477A], and G4 [G721A] mutations were observed from 1, 4, and 1 out of 12 ewes, respectively. Though all 3 mutations were previously reported, this is the first report on their presence in Iranian breeds. The first and second mutations do not alter the amino acids, while G4 is a non-conservative mutation leading to E241K in the prohormone
Conclusion: As the G4 mutation was observed only in ovaries defined superficially as top quality, it could be considered as one of reasons for higher ovulation rate in some sheep. Furthermore since multiple mutations were observed in some cases, it might be possible that combinations of minor mutations in GDF9 and BMP15 interact to affect fecundity in some Iranian sheep breeds