Introduction: 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.Materials and methods: 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.Results and discussion: 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.Conclusions: 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.

Background & aim: Nowadays, most of gene therapy protocols are performed by lentiviral vectors. One of the most important factors which is involved in pancreas development and transcription of insulin gene is pancreatic & duodenal homeobox 1 (PDX-1) transcription factor. The goal of this study was to optimize a lentiviral construct, containing pdx-1 gene, to transfect stem cells towards gene therapy of type-1 diabetes.Methods: In this experimental study, first, the pdx-1 gene was multiplied by PCR from pcDNA3.1- pdx-1 and cloned into pTG19-T vector. Then, pdx-1 was subcloned on upstream of IRES-EGFP gene into IRES2-EGFP vector. At the next step, the cloned parts of IRES-EGFP and pdx-1 were isolated and cloned into the lentiviral expression vector pSINTREM in upstream of TRE-CMV gene.After sequencing, final construct was transfected into HEK 293 cells and gene expression of pdx-1 was evaluated using flow cytometry analysis and reverse fluorescent microscopy.Results: Flow cytometry results and inverted fluorescent microscopy observing showed that pdx-1 and GFP genes are expressed in cells transfected with final recombinant construct.Conclusion: Regarding the design of this construct, to ensure long time expression with higher in vivo and in vitro expression efficiency for stem cells and also use of Tet on induced optimized system, it seems that the current construct can be among the best ones to transfect stem cells.

Bacteriophage lambda comprises critical advantages that have made it as an ideal gene delivery vehicle into the eukaryotic cells. These advantages reside in both the structure and biology of the lambda phage, for example, recent investigations have revealed common ancestry for the tailed phages and eukaryotic DNA viruses. Considering these features as well as the double-stranded nature of its genome, bacteriophage lambda might comprise a great advantage for phage-mediated gene delivery into eukaryotic cells. A phage lambda-derived gene nanocarrier bearing reporter gene GFP (l-GFP) was constructed and utilized to transfect AGS cell line. l-GFP particle-mediated gene delivery and expression as well as its internalization by AGS cell line was investigated.

Background: Leishmaniasis is a neglected disease affecting millions of people worldwide. The treatment of the disease is hampered due to high cost, toxicity and the crisis of drug resistance. Polytope approaches of genetic immunization could be a strategy for prevention of infectious diseases. Furthermore, the identification of Leishmania genome sequence and the application of bioinformatics assist us to devise an effective vaccine’ s candidate. Methods: A linear sequence from predicted epitopes of GP63, LACK and CPC antigens was designed and was optimized using online available algorithms. The synthesized sequence (LAKJB93) was ligated to pEGFP-N1 plasmid. Results: The 264bp sequence was cloned at N terminal of GFP into pEGFP_N1 expression vector and transfect into CHO cell line. Construct was efficient expressed in CHO cells. Conclusion: The protein of LAKJB93 cosnstruct was expressed in CHO cells successfully.

Introduction: Multiple sclerosis (MS) is a disease of the immune system: it attacks the myelin around the axons and leaves them exposed. Destruction of myelin weakens the electrical conduction of ions and thus leads to a lack of communication in the nervous system.Methods: In the present study, we constructed recombinant plasmid and then transformed to E.coli cell. The colonies containing plasmid were selected by Colony PCR. Enzyme digestion and sequencing were utilized to approve the accuracy of the extracted plasmid of these clones. Recombinant plasmid transfect in to mesanchymal stem cells.Results: Plasmid was verification correctly. After transfection, the transcription of MOG gene and the expression of MOG protein were proved by RT-PCR, western blotting and Elisa.Conclusion: Plasmid was constructed correctly and mesenchyme stem cells were successfully transfected by transfection and protein can be expressed well, setting a proper foundation for the future studies on the transplantation of gene modification mesanchymal stem cells in order to promote Multiple sclerosis.

Context: One of the most common aggressive and primary brain tumors is glioma. The majority of diagnoses are referred to highgrade malignant glioblastoma, which carries the worst prognosis. Still, treatment of brain tumors remains a big challenge for clinicians. This study was designed to investigate the efficacy of gene therapy in the treatment of brain cancer. Methods: Studies use genes as a therapeutic agent in brain cancer treatment even alone or in combination with other treatment methods. Full-text papers, which met the inclusion criteria, were independently assessed by two reviewers. Disagreements were resolved by consultation with a third reviewer. Results: Statistical analysis showed that 50% of the papers used a virus, 36% used polymers, and 14% used cells as carriers to transfect the genes as a therapeutic agent in brain tumor models. Data showed that the estimated size of the brain tumor was reduced by using co-treatment of the gene with one of the conventional therapies. Conclusions: According to the results, co-treatment of the gene with conventional therapies could be more effective than the monotherapy methods.

Background: Cutaneous leishmaniasis (CL) is a serious public health problem in many tropical countries. The infection is caused by a protozoan parasite of Leishmania genus transmitted by Phlebotominae sandflies. In the present study, we constructed a eukaryotic expression vector to produce a fusion protein containing LmSTI1 from Leishmania major (L. major) and PpSP42 from Phlebotomus papatasi (Ph. papatasi). In future studies we will test this construct as a DNA vaccine against zoonotic CL. Methods: The nucleotide sequences encoding the LmSTI1 protein and a fragment encoding 79% of PpSP42 were amplified using L. major and Ph. papatasi genomic DNA, respectively. The amplicons were cloned into the pcDNA3. 1(+) eukaryotic expression vector. The recombinant plasmid pcDNA-LmSTI1Pp42 was propagated in Escherichia coli (E. coli) and used to transfect HEK-293T cells. The expressed fusion protein was analyzed by Western blotting using anti-LmSTI1 mouse serum. Results: Sequences encoding LmSTI1 and partial PpSP42 were cloned into pcDNA3. 1(+). Production of the recombinant LmSTI1Pp42 fusion protein was confirmed by Western blotting. Conclusions: An LmSTI1Pp42 fusion protein was expressed in HEK-293T cells. This construct may be an effective DNA vaccine against CL.

Background: Hepatitis B Virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein transactivated by HBV DNA polymerase, screened by suppression subtractive hybridization technique (GenBank accession no: AY450389). The biological function of HBVDNAPTP1 was investigated in this study.Methods: We constructed a vector pcDNA3.1 (-) /myc-His A-HBVDNAPTP1 and used it to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by western blot analysis in the cells. A cDNA library of genes transactivated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using suppression subtractive hybridization (SSH). The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank.Results: Some sequences, such as CIP4, might be involved in apoptosis development. mRNA and protein expression of CIP4 was identified by Real time RT-PCR and western blot in THP-1 cells. HBVDNAPTP1 could down-regulate the expression of CIP4 at both transcription and translation levels.Conclusion: HBVDNAPTP1 may be involved in the positive regulation on the initiation of monocyte apoptosis. The result contribute to reveal the HBVDNAPTP1 biological functions and provide new evidences for further exploration of the regulatory mechanism of HBVDNAPTP1.