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1.
IJFS-International Journal of Fertility and Sterility. 2017; 11 (2): 93-98
in English | IMEMR | ID: emr-186835

ABSTRACT

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

2.
IJRM-Iranian Journal of Reproductive Medicine. 2016; 14 (1): 15-22
in English | IMEMR | ID: emr-177519

ABSTRACT

Background: KLC3 protein as a member of the kinesin light-chain protein family plays an important role in spermatogenesis, during formation of mitochondrial sheath in the mid piece of the sperm tail


Objective:This study for the first time aims to compare the expression of the KLC3 gene between fertile and infertile individuals


Materials and Methods:Semen samples were collected from 19 fertile individuals who were selected from embryo-donor volunteers and 57 infertile individuals who had abnormal sperm parameters according to world health organization criteria. Sperm parameters using computer assisted sperm analysis and the quantitative KLC3-gene expression using the real-time PCR method were measured


Results: Our results revealed a significant correlations between sperm concentration with relative expression of KLC3 only in infertile groups [r=0.45, p=0.00]. A significant correlation was not found between KLC3 expression and sperm motility; however, the relative expression of KLC3 was significantly higher in asthenozoospermic compared to non-asthenozoospermic individuals


Conclusion: Low expression of KLC3 may result in improper function of midpiece, which has important function in sperm motility. The results of this study show that aberrant expression of KLC3 might be associated with phenomena like oligozoospermia and asthenozoospermia. This article is extracted from student's thesis

3.
Cell Journal [Yakhteh]. 2016; 17 (4): 648-658
in English | IMEMR | ID: emr-179293

ABSTRACT

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

4.
Cell Journal [Yakhteh]. 2016; 18 (2): 197-204
in English | IMEMR | ID: emr-183009

ABSTRACT

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

5.
Cell Journal [Yakhteh]. 2016; 18 (2): 221-228
in English | IMEMR | ID: emr-183012

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

6.
IJFS-International Journal of Fertility and Sterility. 2016; 10 (2): 196-207
in English | IMEMR | ID: emr-183072

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

7.
IJFS-International Journal of Fertility and Sterility. 2016; 10 (2): 253-260
in English | IMEMR | ID: emr-183079

ABSTRACT

Background: Selection of sperm for intracytoplasmic sperm injection [ICSI] is usually considered as the ultimate technique to alleviate male-factor infertility. In routine ICSI, selection is based on morphology and viability which does not necessarily preclude the chance injection of DNA-damaged or apoptotic sperm into the oocyte. Sperm with high negative surface electrical charge, named [Zeta potential], are mature and more likely to have intact chromatin. In addition, X-bearing spermatozoa carry more negative charge. Therefore, we aimed to compare the clinical outcomes of Zeta procedure with routine sperm selection in infertile men candidate for ICSI


Materials and Methods: From a total of 203 ICSI cycles studied, 101 cycles were allocated to density gradient centrifugation [DGC]/Zeta group and the remaining 102 were included in the DGC group in this prospective study. Clinical outcomes were com- pared between the two groups. The ratios of Xand Y bearing sperm were assessed by fluorescence in situ hybridization [FISH] and quantitative polymerase chain reaction [qPCR] methods in 17 independent semen samples


Results: In the present double-blind randomized clinical trial, a significant increase in top quality embryos and pregnancy rate were observed in DGC/Zeta group compared to DGC group. Moreover, sex ratio [XY/XX] at birth significantly was lower in the DGC/Zeta group compared to DGC group despite similar ratio of X/Y bearings sper- matozoa following Zeta selection


Conclusion: Zeta method not only improves the percentage of top embryo quality and pregnancy outcome but also alters the sex ratio compared to the conventional DGC method, despite no significant change in the ratio of Xand Ybearing sperm population

8.
Cell Journal [Yakhteh]. 2016; 18 (3): 371-380
in English | IMEMR | ID: emr-183772

ABSTRACT

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

9.
Cell Journal [Yakhteh]. 2016; 18 (3): 438-445
in English | IMEMR | ID: emr-183779

ABSTRACT

Objective: globozoospermia is a rare type of teratozoospermia with incidence of 0.1% among infertile individuals. Phospholipase C zeta [PLC[zeta]] and postacrosomal sheath WW domain binding protein [PAWP] are the main candidates in sperm taking responsibility for oocyte activation during fertilization. Therefore, we aimed to evaluate the expression of these two genes at RNA and protein levels in globozoospermic individuals and compare the results with fertile individuals


Materials and Methods: in this experimental study, semen samples of 21 infertile men with globozoospermia and 25 fertile men were collected. Expression of PLC[zeta] and PAWP at RNA and protein levels were assessed and compared between two groups by quantitative real time polymerase chain reaction [qPCR] and Western blot, respectively


Results: expression of both PLC[zeta] and PAWP were significantly reduced at RNA and protein levels in infertile men with globozoospermia compared to fertile men


Conclusion: this is the first study that simultaneously assessing the respective factors in a large population of globozoospermia, suggested that intra-cytoplasmic sperm injection [ICSI] along with artificial oocyte activation may rescue failed fertilization in routine ICSI

10.
IJFS-International Journal of Fertility and Sterility. 2016; 10 (1): 94-104
in English | IMEMR | ID: emr-178872

ABSTRACT

Background: Personality traits affect human relationships, social interactions, treatment procedures, and essentially all human activities. The purpose of this study is to investigate the personality traits -including sensation seeking, flexibility, and happiness - among a variety of infertile women who were apt to choose assisted reproductive technology [ART] or surrogacy


Materials and Methods: This is a cross-sectional study that was performed on 251 infertile women who visited Isfahan and Tehran Reproductive Medicine Center. These fertility clinics are located in Isfahan and Tehran, Iran. In this study, 201 infertile women who underwent treatment using ART and 50 infertile women who tended to have surrogacy were chosen by convenience sampling. Zuckerman's Sensation Seeking Scale Form V [SSS-V], Psychological Flexibility Questionnaire [adapted from NEO Personality Inventory-Revised] and Oxford Happiness Questionnaire [OHQ] were used as research instruments. All participants had to complete the research instruments in order to be included in this study. Data were analyzed by descriptive-analytical statistics and statistical tests including multivariate analysis of variance [MANOVA] and Z Fisher. Statistically significant effects were accepted for P<0.05


Results: In the sensation-seeking variable, there was a meaningful difference between under-study groups. However, the flexibility and happiness variables did not have a significant difference between under-study groups [P<0.001]. Interaction between education, employment, and financial status was effective in happiness of infertile women underwent ART [P<0.05], while age, education and financial status were also effective in happiness of infertile women sought surrogacy [P<0.05]. A positive meaningful relationship was seen between sensation seeking and flexibility variables in both groups [P<0.05]. And a negative meaningful relationship was seen between sensation seeking and happiness in infertile women who sought surrogacy [P<0.05]. The difference in rate of relationship between sensation seeking and flexibility was meaningful in infertile women who sought either ART or surrogacy [P<0.05]


Conclusion: Sensations seeking as a personality trait is lower in infertile women who underwent treatment using ART compared women who tended to have surrogacy. This study shows that demographic variables are effective in happiness of infertile women. Also, there is a significant relation among sensation seeking, flexibility and happiness in infertile women


Subject(s)
Humans , Women , Middle Aged , Adult , Personality , Pliability , Happiness , Cross-Sectional Studies , Reproductive Techniques, Assisted , Sensation
11.
Cell Journal [Yakhteh]. 2015; 17 (1): 37-48
in English | IMEMR | ID: emr-161616

ABSTRACT

The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells [NCCs]. These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. In this experimental study, we cultured human embryonic stem cells [hESCs] on stromal stem cells from human exfoliated deciduous teeth [SHED] for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells [NPCs] and NCCs. In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity [SDIA] and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development

12.
IJFS-International Journal of Fertility and Sterility. 2014; 8 (1): 21-28
in English | IMEMR | ID: emr-157592

ABSTRACT

The intra-cytoplasmic sperm injection [ICSI] technique selects sperm according to morphology and motility. However, these parameters cannot predict the chromatin integrity of sperm. Considering the detrimental effects of DNA-damaged sperm on reproductive outcomes, novel sperm selection procedures have been proposed to circumvent the possibility of inseminating DNA-damaged sperm. It has been shown that different potential hypo-osmotic swelling test [HOST] patterns possess the potential to differentiate between sperm that have intact or damaged chromatin. Therefore, for the first time, this preliminary study evaluates the role of HOST as a sperm selection procedure in a clinical setting. In this preliminary prospective clinical trial study, we divided infertile couples diagnosed with male infertility into two groups. In the treatment group [n=39], half of the oocytes were inseminated by sperm selected following density gradient centrifugation [DGC group]. The remaining oocytes from the treatment group were inseminated by sperm chosen according to HOST pattern [c, d or e] following DGC processing [HOST group]. In the control group [n=63], all oocytes were inseminated by sperm chosen after DGC. There was a significantly higher percentage of embryos that had good quality, implantation, and chemical pregnancy rates in the HOST group compared to the DGC group [p

Subject(s)
Humans , Male , Female , Chromatin , DNA Fragmentation , Oocytes , Pregnancy Rate , Centrifugation, Density Gradient , Spermatozoa/cytology , Prospective Studies , Embryonic Structures
13.
AJMB-Avicenna Journal of Medical Biotechnology. 2014; 6 (2): 119-122
in English | IMEMR | ID: emr-142234

ABSTRACT

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

14.
IJB-Iranian Journal of Biotechnology. 2014; 12 (4): 1-9
in English | IMEMR | ID: emr-171398

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

16.
IJFS-International Journal of Fertility and Sterility. 2014; 8 (3): 315-324
in English | IMEMR | ID: emr-148947

ABSTRACT

This study examined the causal model of relation between marital relationship status, happiness, and mental health in infertile individuals. In this descriptive study, 155 subjects [men: 52 and women: 78], who had been visited in one of the infertility Centers, voluntarily participated in a self-evaluation. Golombok Rust Inventory of Marital Status, Oxford Happiness Questionnaire, and General Health Questionnaire were used as instruments of the study. Data was analyzed by SPSS17 and Amos 5 software using descriptive statistics, independent sample t test, and path analysis. Disregarding the gender factor, marital relationship status was directly related to happiness [p<0.05] and happiness was directly related to mental health, [p<0.05]. Also, indirect relation between marital relationship status and mental health was significant [p<0.05]. These results were confirmed in women participants but in men participants only the direct relation between happiness and mental health was significant [p<0.05]. Based on goodness of model fit in fitness indexes, happiness had a mediator role in relation between marital relationship status and mental health in infertile individuals disregarding the gender factor. Also, considering the gender factor, only in infertile women, marital relationship status can directly and indirectly affect happiness and mental health


Subject(s)
Humans , Male , Female , Marriage , Happiness , Mental Health , Surveys and Questionnaires
17.
IJPM-International Journal of Preventive Medicine. 2013; 4 (11): 1243-1250
in English | IMEMR | ID: emr-143083

ABSTRACT

Close interaction between retinal pigment epithelium [RPE] and photoreceptors plays an essential role in visual function. The objective of this study is to determine the effects of RPE cells in the differentiation of progenitor derived human embryonic stem cells [hESC] into retinal cells; we developed in vitro co-culture models and compare these models to investigate in which model the expression of photoreceptor markers is superior. It seems the effects of RPE cells on differentiation of retinal progenitor cells [RPCs] through the cell-to-cell contact or with the use of insert and compare of these methods has not been reported yet. Initially, retinal progenitors [RPs] were differentiated from hESC. After isolation of RPE sheet from rabbit eyes, demonstrated these cells maintains the integrity and feature after 2 weeks. Next, we examined the induction of photoreceptors by the co-culture of RPE through insert in 1 week and 2 weeks [indirect] or without insert by the cell-to-cell contact [direct]. The differentiation of retinal cells was verified by protein and gene expression in these three methods. The adherent cells were morphologically examined using phase contrast microscopy and characterized by immunofluorescent staining and reverse transcription polymerase chain reaction [RT PCR]. Evaluation of immunostaining showed that hESC, highly [>80%] can be directed to the RPs fate. Upon co-culture of RPCs with RPE sheet using insert for 2 weeks or by the cell-to-cell contact, these cells differentiated to neural retina and expressed photoreceptor specific markers. However, in direct co-culture, some mature photoreceptor markers like arrestin expressed in compare with indirect co-culture. The expression of late photoreceptor marker could be improved when RPE cells seeded on RPCs in compare with the use of insert.


Subject(s)
Cell Differentiation , Photoreceptor Cells, Vertebrate , Gene Expression , Embryonic Stem Cells , Evaluation Studies as Topic , Reverse Transcriptase Polymerase Chain Reaction , Microscopy, Electron, Scanning Transmission , Coculture Techniques
18.
AJMB-Avicenna Journal of Medical Biotechnology. 2013; 5 (1): 2-9
in English | IMEMR | ID: emr-127550

ABSTRACT

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 , Mice
19.
Cell Journal [Yakhteh]. 2013; 14 (4): 264-269
in English | IMEMR | ID: emr-140460

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 Reaction
20.
AJMB-Avicenna Journal of Medical Biotechnology. 2012; 4 (4): 160-169
in English | IMEMR | ID: emr-151641

ABSTRACT

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

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