m. hajian

The CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated Protein9) method can create a nucleotide sequence complementary to the target sequence in the desired gene by Guide-RNA (gRNA) together with the Cas9 protein, which is a cutting enzyme for cutting in the both DNA strands of the desired sequence accurately and clearly. The DAZL (Deleted in azoospermia-like) gene encodes potential RNA-binding proteins that are expressed in male and female germ cells before and after birth. DAZL, which acts by post-transcriptionally binding mRNA in 3' untranslated regions, regulates the germ cell cycle. DAZL initiates the sexual differentiation of embryonic germ cells. The transfer and transplantation of gene-edited germ cells into recipient males is an effective method for targeted mutagenesis engineering. In mice knocked out for the Dazl gene, the number of testicular stem cells was reduced and it was found that the DAZL gene plays an important role in the differentiation of spermatogonial cells. The purpose of the current research is to edit the DAZL gene by knocking it out using the CRISPR/Cas9 technique and transferring the somatic cell nucleus into the genome of the Bakhtiari goat embryo. The inactivation of the gene will be investigated both at the level of the embryonic cell and the resulting embryo. There are no reports on the production of Bakhtiari goat cells and embryos edited for the DAZL gene by CRISPR technique and somatic nuclear transfer to improve any traits, including reproductive ones. To target the DAZL gene and explore (predict) potential off-target genomic sites, guide RNA (20 bp sequences) immediately upstream of each 5′-NGG in the DAZL gene was designed using the CHOOPCHOOP tool. Plasmid pX459 (9151 bp) was used to insert the gRNA sequence into the CAS9 vector and determine the characteristics and replication of the vector. This plasmid encodes the Cas9 protein along with the puromycin resistance gene under the promoter/enhancer, CAGGS, as well as the gRNA scaffold under the U6 promoter. Plasmid pX459 was digested by BbsI-HF (NEB #R3539) at 37°C for 10 min, followed by purification by NucleoSpin Gel and PCR Clean-up Midi kit (#740,986.20, Machery/Nagel). The purified piece was kept at -20°C for later use. Ligation of oligoduplex carrying diluted gRNA sequence (1:20 ratio from 10 μM source) (1 μL), digested pX459 vector (50 ng), 10x T4DNA ligase buffer (2 μL), and T4DNA ligase (1 μL) in the final concentration of 20 μL of the reaction was carried out. Ligation mixture transformation was performed with NEB 5-alpha Competent E. coli (#C2987I) and then placed on agar culture medium with 100 μg/mL ampicillin and incubated at 37°C overnight. From the cultured plate, five colonies were selected and each was cultured in LB culture medium, followed by miniprep plasmid extraction (Genejet Plasmid miniPrep kit, #K0502). Plasmids carrying the CRISPR system were transferred into cells through an electroporation system. The present results proved the possibility of knocking out the DZAL gene using the CRISPR/Cas9 technique in both fibroblast cell lines and Bakhtiari goat embryos. We performed somatic cell nuclear transfer (SCNT) to examine the embryonic developmental capacity to transition to the cleavage and blastocyst stages. The embryos created by knocking out the DAZL gene could grow and develop. The sequencing analysis using DECODR (Deconvolution of Complex DNA Repair) software showed the knock-out rate of the DAZL gene in fibroblast cells was 83.3% and in cloned blastocysts, it was 55.3%. The rate of blastocyst formation resulting from the cloning process with knockout cells was not significantly different from the control group. Based on these studies, we decided to edit the DAZL gene to produce knockout goat embryos by SCNT. The embryos were examined and evaluated with the help of PCR-RFLP test and sequencing. It turned out that in the first iteration, a knockout of 60.66% (the first ten embryos were paid), and in the second iteration, a knockout of 49.99% (the second ten embryos were paid) were achieved. Such mutations have not been described or detected in Iranian Bakhtiari goats, although polymorphisms have been identified. Using genetic sequencing, the mutations were of the INDEL type and were all in the form of frameshift, which resulted in a change in the protein. The CRISPR/Cas9 system easily influences the desired gene and can be used as a strategy to produce livestock animals that are superior in milk and meat production, reproduction, quality, disease resistance, etc. This research demonstrated the possibility of gene editing using the CRISPR/Cas9 technique in fibroblast cell lines and Bakhtiari goat embryos. The blastocytes produced can be used for transfer to the recipient animal and production of DAZL gene knockout goats for further study and optimization of spermatogonial stem cell transplantation.

Vitrification affects intracellular calcium, fertilization ability, and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether reducing calcium of vitrification solution could improve the fertilization and developmental competence of ovine oocytes.

COCs were collected from the ovine ovary. MII oocytes were divided into 5 groups, one non-vitrified (control) and four vitrified groups 24 hours after COC culture. Vitrified groups were designed according to the presence or absence of EGTA (a calcium chelator) and/or calcium in base media, including mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). Fertilization rate and in vitro development were evaluated after embryo thawing. Also, blastocyst quality was assessed using differential staining. Data analysis was carried out using one-way analysis variance.

There was no significant difference in the viability rate between vitrified groups. Fertilization and the developmental rate decreased in the presence of calcium (p<0.05) but in the calcium-free medium with the EGTA supplementation group, the developmental rate obviously increased. On the other hand, blastocyst cell count in the control group was similar to vitrified groups.

Using a calcium-free cryoprotectant by adding EGTA can improve the quality of vitrified-thawed ovine MII oocyte and also a higher developmental rate in obtained embryos.

According to the development of higher education, the expansion of universities, cultural improvement, more attention women rights and their willing to go to universities, have increased. On the other hand about 60%of total Iranian studentpopulation are studying in human sciences so they need to work. Therefore, the issue of employment status of graduated people is very important. The aim of this study is to investigate the employment and unemployment status of graduated people who have completed their studies on the field of psychology, educational sciences,counseling and library of Al-zahrauniversity. This research is casual comparative. Its data has been gained from Researcher-made questionnaire, Spector’s Job Locus of Control and Sherrer’ General Self Efficacy Inventories were employed as research tools. Descriptive and inferential statistics (independent t-test, k-square, single variable regression) were used to analysis the data. Out of 1320 research population, 130 was randomly selected as a sample group who graduated between 1995-2007. Results showed that Tehrani angraduated found jobs more than others and 77.2% found jobs related to their education.There was not found significant difference between social and economical factorswhich was effective on the labor market, but there was found significant difference between cultural and psychological factors. Job control was predicted byself efficiency. In order to improve Job control and self efficiency of graduates regarding their cultural attitudes to the labor market, suitable trainings like job preparation and entrepreneurship are suggested by researchers.

surface run off from different levels of a land carry many types of impurities and so it should be purified before any consumption. To separate these materials three processes can be used: sedimentation, filtration and flotation. The objective of this study is comparing the flotation and conventional systems in removed turbidity. This is an experimental - intervention in a pilot scale study that compare the turbidity, alkalinity, temperature, pH and TSS in conventional sedimentation system with those of dissolved air flotation. Method in this study, poly aluminum chloride used as a coagulants and kaolin was used to produce turbidity. The results showed that was turbidity removal efficiency of dissolved air flotation system in turbidities NTU20, NTU50-30 and NTU110-90,were respectively 14.7, 11.1 and 10.9 percent higher than those of the conventional deposition system, determined of the level of (p<0.01). According to these results to remove turbidity by dissolved air flotation system then is more effectively to common sedimentation. System Using this system for turbidity removal in a water treatment plant with a relative turbidity from stormy areas that often receives turbid water, is recommended.