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Editorial 2 2 Iran has taken significant steps in the fields of animal cloning, stem cell therapy and recombinant protein drugs in the past decade. The cloning of animals, stem cell therapies and transplantation by the Iranian stem cell transplantation research centers and production of a few recombinant proteins by the Iranian biotech companies and academic institutes is clearly positive for the development of advanced technologies in the field of medical biotechnology. According to the most recent reports in the media, recombinant proteins such as blood coagulation factors VII and VIII are in the process of being manufactured. These are certainly quite significant steps in the development process of recombinant protein drugs. As these recombinant protein drugs come to the Iranian market in the future, it is reasonable to expect prospective studies are designed in order to evaluate the effectiveness and side effects of these drugs in large populations. It is hoped to see a sustained support and a stable environment for the biotech industry in Iran for the continued production of recombinant protein drugs to not only meet the needs of domestic market but also find its way into the international markets in the near future. 1 1 Ali M. Ardekani Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Editorial 169.pdf ####

Constructing a Mouse Oct4 Promoter/EGFP Vector, as a Whole-Cellular Reporter to Monitor the Pluripotent State of Cells 2 2 Background: 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 system is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. Methods: 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. Results: 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. Conclusion: 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. 2 9 Reza Ghorbani Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECRDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECRDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan IranIran Abdolrahman Emamzadeh Department of Biology, Faculty of Sciences, University of Isfahan Department of Biology, Faculty of Sciences, University of Isfahan Iran Yahya Khazaie Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Kianoush Dormiani Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Kamran Ghaedi Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECRDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of IsfahanDepartment of Biology, Faculty of Sciences, University of IsfahanDepartment of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECRDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of IsfahanDepartment of Biology, Faculty of Sciences, University of IsfahanDepartment of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR IranIranIranIran Mohammad Rabbani Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of IsfahanDepartment of Biology, Faculty of Sciences, University of Isfahan Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of IsfahanDepartment of Biology, Faculty of Sciences, University of Isfahan IranIran Mahboubeh Foruzanfar Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Khadijeh Karbalaie Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Fereshteh Karamali Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Liana Lachinani Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Abbas Kiani-Esfahani Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Marzieh Nematollahi Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Iran Mohammad-Hossein Nasr-Esfahani Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECRDepartment of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR Department of Molecular Biotechnology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECRDepartment of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Biotechnology, ACECR IranIran Embryonic stem cellsTranscription factorsMiceEnhanced green fluorescent protein 103.pdf Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126(4): 663-676.##Stadtfeld M, Hochedlinger K. Induced pluripotency: history, mechanisms, and applications. Genes Dev 2010;24(20):2239-2263.##Smith K, Dalton S. Myc transcription factors: key regulators behind establishment and maintenance of pluripotency. Regen Med 2010;5(6):947-959.##Kim JB, Sebastiano V, Wu G, Araúzo-Bravo M J, Sasse P, Gentile L, et al. Oct4-induced pluripotency in adult neural stem cells. Cell 2009;136(3):411-419.##Latchman DS. POU family transcription factors in the nervous system. J Cell Physiol 1999;179(2):126-133.##Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby P, et al. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 1990;345(6277):686-692.##Rappolee D, Basilico C, Patel Y, Werb Z. Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development 1994;120(8):2259-2269.##Berg JS, Goodell MA. An argument against a role for Oct4 in somatic stem cells. Cell Stem Cell 2007;1(4):359-360.##Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24(4):372-376.##Lee KL, Keat Lim SK, Orlov YL, Yit LY, Yang H, Ang LT, et al. Graded nodal/activin signaling titrates conversion of quantitative phospho-smad2 levels into qualitative embryonic stem cell fate decisions. PLoS Genetics 2011;7(6):e1002130.##Blandino G, Deppert W, Hainaut P, Levine A, Lo-zano G, Olivier M, et al . Mutant p53 protein, master regulator of human malignancies: a report on the fifth mutant p53 workshop. Cell Death Differ 2012;19(1):180-183.##Vaccarino FM, Urban AE, Stevens HE, Szekely A, Abyzov A, Grigorenko EL, et al. Annual research review: The promise of stem cell research for neuropsychiatric disorders. J Child Psychol Psychiatry 2011;52(4):504-516.##Soto-Gutierrez A, Tafaleng E, Kelly V, Roy-Chowdhury J, Fox IJ. Modeling and therapy of human liver diseases using induced pluripotent stem cells: How far have we come? Hepatology 2011;53(2):708-711. ##Nordhoff V, Hübner K, Bauer A, Orlova I, Malapetsa A, Schöler HR. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm Genome 2001;12(4):309-317.##Yang HM, Do HJ, Oh JH, Kim JH, Choi SY, Cha KY, et al. Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter. J Cell Biochem 2005;96(4):821-830.##Simonsson S, Gurdon J. DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nat Cell Biol 2004;6(10):984-990. ##Pesce M, Schöler HR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 2001;19(4):271-278.##Ono M, Kajitani T, Uchida H, Arase T, Oda H, Ni-shikawa-Uchida S, et al. OCT4 expression in human uterine myometrial stem/progenitor cells. Hum Reprod 2010;25(8):2059-2067. ##He W, Li K, Wang F, Qin YR, Fan QX. Expression of Oct-4 in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis. World J Gastroenterol 2012;18(7):712-719.##Linn DE, Yang X, Sun F, Xie Y, Chen H, Jiang R, et al. A role for OCT4 in tumor initiation of drug-resistant prostate cancer cells. Genes Cancer 2010;1(9):908-916.##Kim JB, Sebastiano V, Wu G, Araúzo-Bravo MJ, Sasse P, Gentile L, et al. Oct4-Induced pluripotency in adult neural stem cells. Cell 2009:136(3):411-419.##Saxe JP, Tomilin A, Schöler HR, Plath K, Huang J. Post-translational regulation of Oct4 transcriptional activity. PLoS ONE 2009:4(2):e4467. ##Chen T, Yuan D, Wei B, Jiang J, Kang J, Ling K, et al. E-cadherin-mediated cell-cell contact is critical for induced pluripotent stem cell generation. Stem Cells 2010;28(8):1315-1325.##Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989;77(1):61-68.##Keravala A, Portlock J, Nash JA, Vitrant DG, Robbins PD, Calos MP. PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints. J Gene Med 2006;8(8):1008-1017.##Baharvand H, Matthaei KI. Culture condition difference for establishment of new embryonic stem cell lines from the C57BL/6 and BALB/c mouse strains. In Vitro Cell Dev Biol Anim 2004;40(3-4):76-81.##Balaguer P, Boussioux AM, Demirpence E, Nicolas JC. Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands. Luminescence 2001;16(2):153-158.##Motoike T, Loughna S, Perens E, Roman B, Liao W, Chau TC, et al. Universal GFP reporter for the study of vascular development. Genesis 2000;28(2):75-81.##Habermann FA, Wuensch A, Sinowatz F, Wolf E. Reporter genes for embryogenesis research in livestock species. Theriogenology 2007;68(Suppl 1):S116-S124.##Chalfie M, Tu Y, G Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science 1994;263(5148):802-805.##

RORC2 Gene Silencing in Human Th17 Cells by siRNA: Design and Evaluation of Highly Efficient siRNA 2 2 Background: 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. Methods: 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. Results: 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. Conclusion: 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. 10 19 Mazdak Ganjalikhani-Hakemi Immunology Department, Faculty of Medicine, Isfahan University of Medical SciencesApplied Physiology Research Center, Faculty of Medicine, Isfahan University of Medical Sciences Immunology Department, Faculty of Medicine, Isfahan University of Medical SciencesApplied Physiology Research Center, Faculty of Medicine, Isfahan University of Medical Sciences IranIran Kamran Ghaedi Biology Department, Faculty of Sciences, University of Isfahan Biology Department, Faculty of Sciences, University of Isfahan Iran Alireza Andalib Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences Iran Vida Homayouni Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences Iran Mohsen Hosseini Epidemiology Department, Faculty of Health, Isfahan University of Medical Sciences Epidemiology Department, Faculty of Health, Isfahan University of Medical Sciences Iran Abbas Rezaei Immunology Department, Faculty of Medicine, Isfahan University of Medical SciencesApplied Physiology Research Center, Faculty of Medicine, Isfahan University of Medical Sciences Immunology Department, Faculty of Medicine, Isfahan University of Medical SciencesApplied Physiology Research Center, Faculty of Medicine, Isfahan University of Medical Sciences IranIran RNAisiRNATh17 cells 104.pdf Thomas M, Greil J, Heidenreich O. Targeting leukemic fusion proteins with small interfering RNAs: recent advances and therapeutic potentials. Acta Pharmacol Sin 2006;27(3):273-281.##McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002;3(10):737-47.##Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003;4(6):457-467.##Hannon GJ. RNA interference. Nature 2002;418(6894):244-251.##Dykxhoorn DM, Lieberman J. Knocking down disease with siRNAs. Cell 2006;126(2):231-235.##Ichim TE, Li M, Qian H, Popov IA, Rycerz K, Zheng X, et al. RNA interference: a potent tool for gene-specific therapeutics. Am J Transplant 2004;4(8):1227-1236.##Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002;26(2):199-213.##Martinez J, Patkaniowska A, Elbashir SM, Harborth J, Hossbach M, Urlaub H, et al. Analysis of mammalian gene function using small interfering RNAs. Nucleic Acids Res Suppl 2003;(3):333.##Thomas T. Mammalian RNA Interference. New York; 2002.##John J R. RNAi as a treatment for HIV-1 infection. Biotechniques 2006;40:25-29.##Schwarz DS, Hutvgner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003;115(2):199-208.##Ichim TE, Popov IA, Riordan NH, Izadi H, Zhong Z, Yijian L, et al. A novel method of modifying immune responses by vaccination with lipiodol-siRNA mixtures. J Transl Med 2006;4:2.##Saetrom P, Snove O, Jr. A comparison of siRNA efficacy predictors. Biochem Biophys Res Commun 2004;321(1):247-253.##Boese Q, Leake D, Reynolds A, Read S, Scaringe SA, Marshall WS, et al. Mechanistic insights aid computational short interfering RNA design. Methods Enzymol 2005;392:73-96.##Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 2004;32(3):936-948.##Naito Y, Yamada T, Matsumiya T, Ui-Tei K, Saigo K, Morishita S. dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Nucleic Acids Res 2005;33(Web Server issue):W589-W591.##Huppi K, Martin SE, Caplen NJ. Defining and assaying RNAi in mammalian cells. Mol Cell 2005;17(1):1-10.##Amarzguioui M, Prydz H. An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun 2004;316(4):1050-1058.##Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res 2004;32(Web Server issue):W124-W129.##Lovett-Racke AE, Cravens PD, Gocke AR, Racke MK, Stuve O. Therapeutic potential of small interfering RNA for central nervous system diseases. Arch Neurol 2005;62(12):1810-1813.##Takasaki S, Kotani S, Konagaya A. Selecting effective siRNA target sequences for mammalian genes. RNA Biol 2005;2(1):21-27.##Amarzguioui M, Lundberg P, Cantin E, Hagstrom J, Behlke MA, Rossi JJ. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc 2006;1(2):508-517.##Ui-Tei K, Naito Y, Saigo K. Essential notes regarding the design of functional siRNAs for efficient mammalian RNAi. J Biomed Biotechnol 2006;2006(4):65052.##Heale BS, Soifer HS, Bowers C, Rossi JJ. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res 2005;33(3):e30.##Takasaki S, Kotani S, Konagaya A. An effective method for selecting siRNA target sequences in mammalian cells. Cell Cycle 2004;3(6):790-795.##Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol 2004;22(3):326-330.##Luo KQ, Chang DC. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem Biophys Res Commun 2004 21;318(1):303-310.##Birmingham A, Anderson E, Sullivan K, Reynolds A, Boese Q, Leake D, et al. A protocol for designing siRNAs with high functionality and specificity. Nat Protoc 2007;2(9):2068-2078.##Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J, Robinson K, et al. Off-target effects by siRNA can induce toxic phenotype. RNA 2006;12(7):1188-1196.##Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, et al. 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 2006;3(3):199-204.##Yamada T, Morishita S. Accelerated off-target search algorithm for siRNA. Bioinformatics 2005;21(8):1316-1324.##Tafer H, Ameres SL, Obernosterer G, Gebeshuber CA, Schroeder R, Martinez J, et al. The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol 2008;26(5):578-583.##volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupe P, Barillot E, et al. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 2008;9(6):650-657.##Ganjalikhani HM, Ghaedi K, Andalib A, Hosseini M, Rezaei A. Optimization of human Th17 cell differentiation in vitro: evaluating different polarizing factors. In Vitro Cell Dev Biol Anim 2011;47:581-592.##Yang L, Anderson DE, Baecher-Allan C, Hastings WD, Bettelli E, Oukka M, et al. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 2008;454(7202):350-352.##Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ddCt method. Methods 2001;25:402-408.##Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29(9):e45.##Muckstein U, Tafer H, Hackermuller J, Bernhart SH, Stadler PF, Hofacker IL. Thermodynamics of RNA-RNA binding. Bioinformatics 2006;22(10):1177-1182.##Ui-Tei K, Naito Y, Zenno S, Nishi K, Yamato K, Takahashi F, et al. Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Res 2008;36(7):2136-2151.##Amarzguioui M, Rossi JJ. Principles of Dicer substrate (D-siRNA) design and function. Methods Mol Biol 2008;442:3-10.##Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J, et al. Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. RNA 2006;12(7):1197-1205.##Snove O, Jr., Nedland M, Fjeldstad SH, Humberset H, Birkeland OR, Grunfeld T, et al. Designing effective siRNAs with off-target control. Biochem Biophys Res Commun 2004;325(3):769-773.##Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell 2003;115(2):209-216.##

Expression, Purification and Characterization of Three Overlapping Immunodominant Recombinant Fragments from Bordetella pertussis Filamentous Hemagglutinin 2 2 Background: Filamentous hemagglutinin (FHA) is one of the most important immunoprotective antigens of Bordetella pertussis (B.pertussis) and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. Methods: Three overlapping coding sequences of FHA antigen were amplified from B.pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli (E. coli) BL21(DE3) strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells (PBMC) proliferation and IFN-γ production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively. Results: Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21(DE3). SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN-γ production. Conclusion: Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immuno-protective. 20 28 Hossein Asgarian-Omran Department of Immunology, School of Medicine, Tehran University of Medical SciencesDepartment of Immunology, School of Public Health, Tehran University of Medical Sciences Department of Immunology, School of Medicine, Tehran University of Medical SciencesDepartment of Immunology, School of Public Health, Tehran University of Medical Sciences IranIran Ali Akbar Amirzargar Department of Immunology, School of Medicine, Tehran University of Medical Sciences Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Mohammad Arjmand Department of Biochemistry, Pasteur Institute of Iran Iran Mohammadreza Eshraghian Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences Iran Behrooz Nikbin Department of Immunology, School of Medicine, Tehran University of Medical Sciences Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Saeid Eshraghi Department of Microbiology, School of Public Health, Tehran University of Medical Sciences Department of Microbiology, School of Public Health, Tehran University of Medical Sciences Iran Marzieh Mahdavi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Jalal Khoshnoodi Department of Immunology, School of Public Health, Tehran University of Medical Sciences Department of Immunology, School of Public Health, Tehran University of Medical Sciences Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Hodjattallah Rabbani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Fazel Shokri Department of Immunology, School of Medicine, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Medicine, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranIran Bordetella pertussisFilamentous hemagglutininImmunodominantProkaryotic expressionRecombinant antigen 105.pdf Crowcroft NS, Pebody RG. Recent developments in pertussis. Lancet 2006;367(9526):1926-1936.##Marzouqi I, Richmond P, Fry S, Wetherall J, Mukkur T. Development of improved vaccines against whooping cough: current status. Hum Vaccin 2010;6(7):543-553.##Berbers GA, de Greeff SC, Mooi FR. Improving pertussis vaccination. Hum Vaccin 2009;5(7):497-503.##Xu Y, Zhang S, Bolgiano B, Tan Y, Asokanathan C, Zhang H, et al. Comparison of recombinant and native pertactin of Bordetella pertussis. Vaccine 2011;29(10):1974-1980.##Higgs R, Higgins SC, Ross PJ, Mills KH. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol 2012;5(5):485-500.##Mazar J, Cotter PA. Topology and maturation of filamentous haemagglutinin suggest a new model for two-partner secretion. Mol Microbiol 2006;62(3):641-654.##Colombi D, Horton DS, Oliveira ML, Sakauchi MA, Ho PL. Antibodies produced against a fragment of filamentous haemagglutinin (FHA) of Bordetella pertussis are able to inhibit hemagglutination induced by the whole adhesin. FEMS Microbiol Lett 2004;240(1):41-47.##Knight JB, Huang YY, Halperin SA, Anderson R, Morris A, Macmillan A, et al. Immunogenicity and protective efficacy of a recombinant filamentous haemagglutinin from Bordetella pertussis. Clin Exp Immunol 2006;144(3):543-551.##Relman D, Tuomanen E, Falkow S, Golenbock DT, Saukkonen K, Wright SD. Recognition of a bacterial adhesion by an integrin: macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 1990;61(7):1375-1382.##Prasad SM, Yin Y, Rodzinski E, Tuomanen EI, Masure HR. Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis. Infect Immun 1993;61(7):2780-2785.##Hannah JH, Menozzi FD, Renauld G, Locht C, Brennan MJ. Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA. Infect Immun 1994;62(11):5010-5019.##Di Tommaso A, Domenighini M, Bugnoli M, Tagliabue A, Rappuoli R, De Magistris MT. Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses. Infect Immun 1991;59(9):3313-3315.##Leininger E, Bowen S, Renauld-Mongenie G, Rouse JH, Menozzi FD, Locht C, et al. Immuno-dominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin. J Infect Dis 1997;175(6):1423-1431.##Del Giudice G, Rappuoli R. Genetically derived toxoids for use as vaccines and adjuvants. Vaccine 1999;17(Suppl 2):S44-52.##Shabani M, Asgarian-Omran H, Hojjat-Farsangi M, Vossough P, Sharifian RA, Toughe GR, et al. Comparative expression profile of orphan receptor tyrosine kinase ROR1 in Iranian patients with lymphoid and myeloid leukemias. Avicenna J Med Biotech 2011;3(3):119-125.##Cherry JD. The present and future control of pertussis. Clin Infect Dis 2010;51(6):663-667.##Nascimento IP, Dias WO, Mazzantini RP, Miyaji EN, Gamberini M, Quintilio W, et al. Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice. Infect Immun 2000;68(9):4877-4883.##Lee SF, Halperin SA, Knight JB, Tait A. Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii. Appl Environ Microbiol 2002;68(9):4253-4258.##Jinyong Z, Xiaoli Z, Weijun Z, Ying G, Gang G, Xuhu M, et al. Fusion expression and immunogenicity of Bordetella pertussis PTS1-FHA protein: implications for the vaccine development. Mol Biol Rep 2011;38(3):1957-1963.##Khafri A, Aghaiypour K, Najar Peerayeh S, Ghorbani R. Cloning and expression of S1 subunit of pertussis toxin in Escherichia coli. Avicenna J Med Biotech 2011;3(1):19-24.##Relman DA, Domenighini M, Tuomanen E, Rappuoli R, Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci USA 1989;86(8):2637-2641.##Delisse-Gathoye AM, Locht C, Jacob F, Raaschou-Nielsen M, Heron I, Ruelle JL, et al. Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis. Infect Immun 1990;58(9):2895-2905.##Renauld-Mongenie G, Cornette J, Mielcarek N, Menozzi FD, Locht C. Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin. J Bacteriol 1996;178(4):1053-1060.##Wilson DR, Siebers A, Finlay BB. Antigenic analysis of Bordetella pertussis filamentous hemag-glutinin with phage display libraries and rabbit anti-filamentous hemagglutinin polyclonal antibodies. Infect Immun 1998;66(10):4884-4894.##Piatti G. Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis. FEMS Immunol Med Microbiol 1999;23(3):235-241.##Alonso S, Reveneau N, Pethe K, Locht C. Eighty-kilodalton N-terminal moiety of Bordetella pertussis filamentous hemagglutinin: adherence, immunogenicity, and protective role. Infect Immun 2002;70(8):4142-4147.##

Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production 2 2 Background: Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Methods: Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3 , the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants Results: Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. Conclusion: A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen. 29 34 Hassan Hamedi Department of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran Department of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran Iran Ali Misaghi Department of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran Department of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran Iran Mohammad Hossein Modarressi Department of Medical Genetics, Tehran University of Medical Sciences Department of Medical Genetics, Tehran University of Medical Sciences Iran Taghi Zahraei Salehi Department of Microbiology, Faculty of Veterinary of Medicine, University of Tehran Department of Microbiology, Faculty of Veterinary of Medicine, University of Tehran Iran Dorsa Khorasanizadeh Fungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran Iran Vahid Khalaj Fungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran Iran ProbioticRecombinant proteinsUracilYeasts 106.pdf D’Silva I. Recombinant technology and probiotics. Int J Eng Technol 2011;3(4):288-293.##Gaon D, Garcia H, Winter L, Rodriguez N, Quintas R, Gonzalez SN, et al. Effect of Lactobacillus strains and Saccharomyces boulardii on persistent diarrhea in children. Medicina 2003;63(4):293-298.##Hebuterne X. Gut changes attributed to ageing: effects on intestinal microflora. Current opinion in clinical nutrition and metabolic care. Curr Opin Clin Nutr Metab Care 2003;6(1):49-54.##Buts JP. Twenty-five years of research on Saccharomyces boulardii trophic effects: updates and perspectives. Digestive diseases and sciences. Dig Dis Sci 2009;54(1):15-18.##Riaz M, Alam S, Malik A, Ali SM. Efficacy and safety of Saccharomyces boulardii in acute childhood diarrhea: A double blind randomised controlled trial. Indian J Pediatr 2012;79(4):478-482.##Czerucka D, Piche T, Rampal P. Review article: yeast as probiotics -- Saccharomyces boulardii. Aliment Pharmacol Ther 2007;26(6):767-778.##Hashimoto S, Ogura M, Aritomi K, Hoshida H, Nishizawa Y, Akada R. Isolation of auxotrophic mutants of diploid industrial yeast strains after UV mutagenesis. Appl Environ Microbiol 2005;71(1):312-319.##Nayak S. Biology of eukaryotic probiotics. In: Liong M (eds). Probiotics. Berlin, Heidelberg: Springer-Verlag; 2011, 29-54.##Pronk JT. Auxotrophic yeast strains in fundamental and applied research. Appl Environ Microbiol 2002;68(5):2095-2100.##Umezu K, Amaya T, Yoshimoto A, Tomita K. Purification and properties of orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase from baker's yeast. J Biochem 1971;70(2):249-262.##Boeke JD, LaCroute F, Fink GR. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 1984;197(2):345-346.##Peter P. Isolation of yeast DNA. In: Evans I (eds). Yeast protocols: Methods in cell and molecular biology. Totowa: Humana Press Inc; 1996, 103-107.##Benatuil L, Perez JM, Belk J, Hsieh CM. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 2010;23(4):155-159.##van der Aa Kuhle A, Skovgaard K, Jespersen L. In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains. Int J Food Microbiol 2005;101(1):29-39.##Sleator RD, Hill C. Patho-biotechnology: using bad bugs to do good things. Curr Opin Biotechnol 2006;17(2):211-216.##Alani E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 1987;116(4):541-545.##van der Aa Kuhle A, Jespersen L. The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene. Syst Appl Microbiol 2003;26(4):564-571.##Sharaf AN, Abosereh NAR, Abdalla SM, HALA Mohamed, Salim RGS. Impact of some genetic treatments on the probiotic activities of Saccharomyces boulardii. Res J Cell Mol Biol 2009;3(1):12-19.##Abosereh NA, HALA Mohamed, ABA El-Chalk A. Genetic construction of potentially probiotic Saccharomyces boulardii yeast strains using intraspecific protoplast fusion. J Appl Sci 2007;3(3):209-217.##

Cloning and Expression of Gumboro VP2 Antigen in Aspergillus niger 2 2 Background: Infectious Bursal Disease Virus (IBDV) causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger (A. niger). Methods: Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG - protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. Results: A number of pyrG+ positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. Conclusion: In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry. 35 41 Mohammad Azizi Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of IranIndustrial and Environmental Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB) IranIran Bagher Yakhchali Industrial and Environmental Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB) Iran Abdolreza Ghamarian Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Somayeh Enayati Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Mahvash Khodabandeh Industrial and Environmental Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB) Iran Vahid Khalaj Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Aspergillus nigerRecombinant proteinsVP2 protein 107.pdf Azad AA, Barrett SA, Fahey KJ. The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus. Virology 1985;143(1):35-44.##International Office of Epizootics (IOE), Biological Standards Commission. Infectious bursal disease (Gumboro disease). In: Commission OBS, ed. Manual of diagnostic tests and vaccines for terrestrial animals: mammals, birds and bees. Paris: OIE; 2008, 549-565.##Mundt E, Beyer J, Muller H. Identification of a novel viral protein in infectious bursal disease virus-infected cells. J Gen Virol 1995;76(Pt 2):437-443.##Berg TP. Acute infectious bursal disease in poultry: a review. Avian Pathol 2000;29(3):175-194.##Berg TP, Gonze M, Morales D, Meulemans G. Acute infectious bursal disease in poultry: Immunological and molecular basis of antigenicity of a highly virulent strain. Avian Pathol 1996;25(4):751-768.##Wu H, Singh NK, Locy RD, Scissum-Gunn K, Giambrone JJ. Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana. Biotechnol Lett 2004;26(10):787-792.##Wu J, Yu L, Li L, Hu J, Zhou J, Zhou X. Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J 2007;5(5):570-578.##Li L, Fang WH, Fan YJ, Xu J, Fang L, Li JR, et al. Expression of the infectious bursal disease virus polyprotein in Vero cells using attenuated Salmonella typhimurium as transgenic carrier. Sheng Wu Gong Cheng Xue Bao 2004;20(3):437-440##Mahmood MS, Hussain I, Siddique M, Akhtar M, Ali S. DNA vaccination with VP2 gene of very virulent infectious bursal disease virus (vvIBDV) delivered by transgenic E. coli DH5alpha given orally confers protective immune responses in chickens. Vaccine 2007;25(44):7629-7635.##Punt PJ, van Biezen N, Conesa A, Albers A, Mangnus J, van den Hondel C. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol 2002;20(5):200-206.##Meyer V, Wu B, Ram AF. Aspergillus as a multi-purpose cell factory: current status and perspectives. Biotechnol Lett 2011;33(3):469-476.##Moore D, Chiu SW. Fungal products as food. In: Pointing S, Hyde K (eds). Bio-exploitation of filamentous fungi. Hong Kong: Fungal Diversity Press; 2001, 223-251.##Jayani R, Saxena S, Gupta R. Microbial pectinolytic enzymes: A review. Process Biochem 2005;40:2931-2944.##Lee K, Lee S, Lee B. Aspergillus oryzae as probiotic in poultry-A review. Int J Poult Sci 2006;5(1):1-3.##Fleissner A, Dersch P. Expression and export: recombinant protein production systems for Aspergillus. Appl Microbiol Biotechnol 2010;87(4):1255-1270.##Aleksenko AY, Clutterbuck AJ. Recombinational stability of replicating plasmids in Aspergillus nidulans during transformation, vegetative growth and sexual reproduction. Curr Genet 1995;28(1):87-93.##Aleksenko A, Clutterbuck AJ. Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements. Fungal Genet Biol 1997;21(3):373-387.##Aleksenko A, Nikolaev I, Vinetski Y, Clutterbuck AJ. Gene expression from replicating plasmids in Aspergillus nidulans. Mol Gen Genet 1996;253(1-2):242-246.##Bos CJ, Debets AJ, Swart K, Huybers A, Kobus G, Slakhorst SM. Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 1988;14(5):437-443.##Vogel H. A convenient growth medium for Neurospora (medium N). Microb Genet Bull 1956;13:42-44.##van Hartingsveldt W, Mattern IE, van Zeijl CM, Pouwels PH, van den Hondel CA. Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 1987;206(1):71-75.##Macreadie IG, Vaughan PR, Chapman AJ, McKern NM, Jagadish MN, Heine HG, et al. Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast. Vaccine 1990;8(6):549-552.##Sharma R, Katoch M, Srivastava P, Qazi GN. Approaches for refining heterologous protein production in filamentous fungi. World J Microbiol Biotechnol 2009;25(12):2083-2094.##Storms R, Zheng Y, Li H, Sillaots S, Martinez-Perez A, Tsang A. Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger. Plasmid 2005;53(3):191-204.##Gouka RJ, Punt PJ, Hessing JG, van den Hondel CA. Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains. Appl Environ Microbiol 1996;62(6):1951-1957.##Koda A, Bogaki T, Minetoki T, Hirotsune M. High expression of a synthetic gene encoding potato alpha-glucan phosphorylase in Aspergillus niger. J Biosci Bioeng 2005;100(5):531-537.##Prathumpai W, Flitter SJ, McIntyre M, Nielsen J. Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus. Appl Microbiol Biotechnol 2004;65(6):714-719.##Heine HG, Hyatt AD, Boyle DB. Modification of infectious bursal disease virus antigen VP2 for cell surface location fails to enhance immunogenicity. Virus Res 1994;32(3):313-328.##Azad AA, Macreadie IGT, Mckern NML, Vaughan PRR, Jagadish MNGP, Fahey KJT, inventors; Production of IBDV VP2 in highly immunogenic form. Australia patent AU PCT/AU 1990/000224. 1997 May 29.##Aziz MA, Midha S, Waheed SM, Bhatnagar R. Oral vaccines: new needs, new possibilities. Bioessays 2007;29(6):591-604.##Levine MM. Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine. BMC Biol 2010;8:129.##Solis de los Santos F, Farnell MB, Tellez G, Balog JM, Anthony NB, Torres-Rodriguez A, et al. Effect of prebiotic on gut development and ascites incidence of broilers reared in a hypoxic environment. Poult Sci 2005;84(7):1092-1100.##Londero A, Menconi A, Reginatto AR, Bacocina AI, Wolfenden A, Shivaramaiah S, et al. Effect of an aspergillus meal prebiotic on salmonella infection in turkeys and broiler chickens. Int J Poult Sci 2011;10(12):946-951.##

Antifungal Indole and Pyrrolidine-2,4-Dione Derivative Peptidomimetic Lead Design Based on In Silico Study of Bioactive Peptide Families 2 2 Background: The rise of opportunistic fungal infections highlights the need for development of new antimicrobial agents. Antimicrobial Peptides (AMPs) and Antifungal Peptides (AFPs) are among the agents with minimal resistance being developed against them, therefore they can be used as structural templates for design of new antimicrobial agents. Methods: In the present study four antifungal peptidomimetic structures named C1 to C4 were designed based on plant defensin of Pisum sativum. Minimum inhibitory concentrations (MICs) for these structures were deter-mined against Aspergillus niger N402, Candida albicans ATCC 10231, and Saccharomyces cerevisiae PTCC 5052. Results: C1 and C2 showed more potent antifungal activity against these fun-gal strains compared to C3 and C4. The structure C2 demonstrated a potent antifungal activity among them and could be used as a template for future study on antifungal peptidomemetics design. Sequences alignments led to identifying antifungal decapeptide (KTCENLADTY) named KTC-Y, which its MIC was determined on fungal protoplast showing 25 (μg/ml) against Asper-gillus fumigatus Af293. Conclusion: The present approach to reach the antifungal molecules seems to be a powerful approach in design of bioactive agents based on AMP mimetic identification. 42 53 Shoeib Moradi Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Parisa Azerang Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Vahid Khalaj Fungal Biotechnology Group, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Soroush Sardari Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Antifungal agentsDefensinsDrug designPeptidomimeticsProtoplasts 108.pdf Hong SY, Park TG, Lee KH. The effect of charge increase on the specificity and activity of a short antimicrobial peptide. Peptides 2001;22(10):1669-1674.##Dhople V, Krukemeyer A, Ramamoorthy A. The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochim Biophys Acta 2006;1758(9):1499-1512.##Brogden KA. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacterial. Nat Rev Microbiol 2005;3(3):238-250.##Ouellet M, Otis F, Voyer N, Auger M. Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes. Insights on their modes of action. Biochim Biophys Acta 2006;1758(9):1235-1244.##Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1(3):156-164.##Amsterdam D. Susceptibility testing of antimicrobials in liquid media. In: Lorian V (eds). Antibiotics in laboratory medicine. Baltimore Md: Williams and Wilkins; 1996, 52-111. ##Lopez SF, Kim HS, Choi EC, Delgado M, Granja JR, Khasanov A, et al. Antibacterial agents based on the cyclic D,L-alpha-peptide architecture. Nature 2001;412(6845):452-455.##Soltani S, Keymanesh K, Sardari S. Evaluation of structural features of membrane acting antifungal peptides by artificial neural networks. J Biol Sci 2008;8(5):834-845.##Chu KT, Xia L, Ng TB. Pleurostrin, an antifungal peptide from the oyster mushroom. Peptides 2005;26(11):2098-2103.##Wong JH, Ng TB. Vulgarinin, a broad-spectrum antifungal peptide from haricot beans (Phaseolus vulgaris). Int J Biochem Cell Biol 2005;37(8):1626-1632.##Ye XY, Ng TB, Rao PF. Cicerin and arietin, novel chickpea peptides with different antifungal potencies. Peptides 2002;23(5):817-822.##Lin P, Xia L, Ng TB. First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species. Peptides 2007;28(8):1514-1519.##Fujimura M, Minami Y, Watanabe K, Tadera K. Purification, characterization, and sequencing of a novel type of antimicrobial peptides, Fa-AMP1 and Fa-AMP2, from seeds of buckwheat (Fagopyrum esculentum Moench). Biosci Biotechnol Biochem 2003;67(8):1636-1642.##Wong JH, Ng TB. Lunatusin, a trypsin-stable antimicrobial peptide from lima beans (Phaseolus lunatus L.). Peptides 2005;26(11):2086-2092.##Iijima R, Kisugi J, Yamazaki M. A novel antimicrobial peptide from the sea hare Dolabella auricularia. Dev Comp Immunol 2003;27(4):305-311.##Ng TB. Peptides and proteins from fungi. Peptides 2004;25(6):1055-1073.##Pelegrini PB, Noronha EF, Muniz MAR, Vasconcelos IM, Chiarello MD, Oliveira JT, et al. An antifungal peptide from passion fruit (Passiflora edulis) seeds with similarities to 2S albumin proteins. Biochim Biophys Acta 2006;1764(6):1141-1146.##Thevissen K, Francois EJA, Sijtsma L, Amerongen A, Schaaper, WMM, Meloen R, et al. Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4. Peptides 2005;26(7):1113-1119.##Wong JH, Ng TB. Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase. Peptides 2005;26(7):1120-1126.##Huang RH, Xiang Y, Liu XZ, Zhang Y, Hu Z, Wang DC. Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv. FEBS Lett 2002;521(1-3):87-90.##Wang HX, Ng TB. Isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds. Peptides 2003;24(7):969-972.##Wang X, Bunkers GJ, Walters MR, Thoma RS. Purification and characterization of three antifungal proteins from cheeseweed (Malva parviflora). Biochem Biophys Res Commun 2001;282(5):1224-1228.##Ngai PHK, Ng TB. Coccinin, an antifungal peptide with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from large scarlet runner beans. Peptides 2004;25(12):2063-2068.##24. Ngai PHK, Zheng Z, Ng TB. Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea. Peptides 2005;26(2):191-196.##Koo JC, Lee SY, Chun HJ, Cheong YH, Choi JS. Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity. Biochimica et Biophysica Acta 1998;1382(1):80-90.##Wang HX, Ng TB. An antifungal peptide from red lentil seeds. Peptides 2007;28(3):547-552.##Wang HX, Ng TB, Liu Q. Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris. Peptides 2004;25(4):693-696.##Ngai PHK, Ng TB. Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells. Life Sci 2003;73(26):3363-3374.##Lam SK, Ng TB. First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeiji) together with evidence for synergism of their antifungal effect. Arch Biochem Biophys 2001;393(2):271-280.##Yang X, Li J, Wang X, Fang W, Bidochka MJ, She R, Xiao Y, et al. Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds. Peptides 2006;27(7):1726-1731.##Ye XY, Ng TB. Peptides from pinto bean and red bean with sequence homology to cowpea 10-kDa protein precursor exhibit antifungal, mitogenic, and HIV-1 reverse transcriptase-inhibitory activities. Biochem Biophys Res Commun 2001;285(2):424-429.##Wang H, Ng TB. Novel antifungal peptides from Ceylon spinach seeds. Biochem Biophys Res Commun 2001;288(4):765-770.##Gao GH, Liu W, Dai JX, Wang JF, Hu Z, Zhang Y, et al. Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana. Biochemistry 2001;40(37):10973-10978.##Skerlavaj B, Benincasa M, Risso A, Zanetti M, Gennaro R. SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Lett 1999;463(1-2):58-62.##Wang H, Ng TB. Isolation of cicadin, a novel and potent antifungal peptide from dried juvenile cicadas. Peptides 2002;23(1):7-11.##Wang HX, Ng TB. Eryngin, a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii. Peptides 2004;25(1):1-5.##Lampert A, Noble C, Davies W, Haefner B, Wilson J. Monitor-biology. Drug Discov Today 2004;9(22):985-987.##Yang WY, Wen SY, Huang YD, Ye MQ, Deng XJ, Han D, et al. Functional divergence of six isoforms of antifungal peptide Drosomycin in Drosophila melanogaster. Gene 2006;379:26-32.##Yamagishi H, Fitzgerald DH, Sein T, Walsh TJ, O'Connell BC. Saliva affects the antifungal activity of exogenously added histatin 3 towards Candida albicans. FEMS Microbiol Lett 2005;244(1):207-212.##Huang X, Xie W, Gong Z. Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba. FEBS Lett 2000;478(1):123-126.##Ahn HS, Cho W, Kang SH, Ko SS, Park MS, Cho H, et al. Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study. Peptides 2006;27(4):640-648.##Simmaco M, Mignogna G, Barra D, Bossa F. Antimicrobial peptides from skin secretions of Rana esculenta. J Biol Chem 1994;269(16):11956-11961.##De Grado, WF, Kezdy FJ, Kaiser ET. Design, synthesis and characterization of a cytotoxic peptide with melittin-like activity. J Am Chem Soc 1981;103:679-681.##Oh JE, Hong SY, Lee KH. The comparison of characteristics between membrane-active antifungal peptide and its pseudopeptides. Bioorg Med Chem 1999;7(11):2509-2515.##Tanaka H, Suzuki K. Expression profiling of a diapause-specific peptide (DSP) of the leaf beetle Gastrophysa atrocyanea and silencing of DSP by double-strand RNA. J Insect Physiol 2005;51(6):701-707.##Wong JH, Ng TB. Gymnin, a potent defensin-like antifungal peptide from the Yunnan bean (Gymnocladus chinensis Baill). Peptides 2003;24(7):963-968.##Jang WS, Kim HK, Lee KY, Kim SA, Han YS, Lee IH. Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium. FEBS Lett 2006;580:1490-1496.##Wang HX, Ng TB. Ascalin, a new anti-fungal peptide with human immunodeficiency virus type 1 reverse transcriptase-inhibiting activity from shallot bulbs. Peptides 2002;23(6):1025-1029.##Wang HX, Ng TB. An antifungal protein from the pea Pisum sativum var. arvense Poir. Peptides 2006;27(7):1732-1737.##Chu KT, Liu KH, Ng TB. Cicerarin, a novel antifungal peptide from the green chickpea. Peptides 2003;24(5):659-663.##Lipkin A, Anisimova V, Nikonorova A, Babakov A, Krause E, Bienert M, et al. An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds. Phytochemistry 2005;66(20):2426-2431.##Xia L, Ng TB. Actinchinin, a novel antifungal protein from the gold kiwi fruit. Peptides 2004;25(7):1093-1098.##EBI: Multiple sequence alignment [Internet]. Cambridge (United Kingdom): European Bioinformatics Institute; c1980. [cited 2012 Nov 6] Available from: http://www.ebi.ac.uk/.##Notredame C, Higgins DG, Heringa J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 2000;302(1):205-217.##EBI: The Basic Local Alignment Search Tool [Internet]. Cambridge (United Kingdom): European Bioinformatics Institute; c1980. [cited 2012 Nov 6] Available from: http:// blast.ncbi.nlm.nih.gov/ Blast.cgi/.##PDB: Protein Data Bank [Internet]. San Diego (CA): San Diego Supercomputer Center (SDSC); 2003. (cited 2012 Nov 6). Available from: http:// www.rcsb.org/pdb/home/home.do/.##Goede A, Michalsky E, Schmidt U, Preissner R. Super mimic-fitting peptide mimetics into protein structures. BMC Bioinformatics 2006;7:11.##Fullbeck M, Michalsky E, Jaeger IS, Henklein P, Kuhn H, Ruck-Braun K, et al. Design and biological evaluation of photo-switchable inhibitors. Genome Information 2006;17(1):141-151.##Romano J, Nimrod G, Ben-Tal N, Shadkchan Y, Baruch K, Sharon H, et al. Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence. Microbiology 2006;152(7):1919-1928.##Cuenca-Estrella M, Lee-Yang W, Ciblak MA, Arthington-Skaggs BA, Mellado E, Warnock DW, et al. Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution procedures for antifungal susceptibility testing of candida species. Antimicrob Agents Chemother 2002;46(11):3644-3647. ##Gomez-Lopez A, Aberkane A, Petrikkou E, Mellado E, Rodriguez-Tudela JL, Cuenca-Estrella M. Analysis of the influence of tween concentration, inoculum size, assay medium, and reading time on susceptibility testing of Aspergillus spp. J Clin Microbiol 2005;43(3):1251-1255.##Swiss-Pdb Viewer [Internet]. Bern (Switzerland): The SIB Swiss Institute of Bioinformatics; 1995-2001. (cited 2012 Nov 6). Available from: http:// www.expasy.ch/spdbv, v3.7 SP5/. ##Enhanced NCI Database Browser [Internet]. Nuremberg, Germany: Frederick National Laboratory for Cancer Research (FNLCR). c1997. (cited 2012 Nov 6) Available from: http://cactus.nci.nih.gov/ ncidb2.##ZINC [Internet]. San Francisco (CA): Shoichet Laboratory at University of California San Francisco (UCSF); 2004. (cited 2012 Nov 6). Available from: http://zinc.docking.org/.##Ruge E, Korting HC, Borelli C. Current state of three-dimensional characterisation of antifungal targets and its use for molecular modelling in drug design. Int J Antimicrob Agents 2005;26(6):427-441.##Stigers KD, Soth MJ, Nowick JS. Designed molecules that fold to mimic protein secondary structures. Curr Opin Chem Biol 1999;3(6):714-723.##Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 2004;5(1):113.##Rosenberg MS. Multiple sequence alignment accuracy and evolutionary distance estimation. BMC Bioinformatics 2005;6(1):278.##Thomma B, Cammue BPA, Thevissen K. Plant defensins. Planta 2002;216(2):193-202.##Jung HJ, Park YK, Sung WS, Suh BK, Lee J, Hahm KS, et al. Fungicidal effect of pleurocidin by membrane-active mechanism and design of enantiomeric analogue for proteolytic resistance. Biochim Biophys Acta 2007;1768(6):1400-1405.##Sung WS, Lee J, Lee DG. Fungicidal effect of piscidin on Candida albicans: pore formation in lipid vesicles and activity in fungal membranes. Biol Pharm Bull 2008;31(10):1906-1910.##Epand RM, Vogel HG. Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1999;1462:11-28.##Saberwal G, Nagaraj R. Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities. Biochim Biophys Acta 1994;1197(2):109-131.##Lawyer C, Pai S, Watabe M, Borgia P, Mashimo T, Eagleton L, et al. Antimicrobial activity of a 13 amino acid tryptophan-rich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides. FEBS Lett 1996;390(1):95-98.##Chan DI, Prenner EJ, Vogel HJ. Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action. Biochim Biophys Acta 2006;1758(9):1184-1202.##Ramamoorthy A, Thennarasu S, Tan A, Lee DK, Clayberger C, Krensky AM. Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin. Biochim Biophys Acta 2006;1758:154-163.##Andreu D, Merrifield RB, Steiner H, Boman HG. N-terminal analogues of cecropin A: synthesis, antibacterial activity, and conformational properties. Biochemistry 1985;24(7):1683-1688.##Xie Y, Kai ZP, Tobe SS, Deng XL, Ling Y, Wu XQ, et al. Design, synthesis and biological activity of peptidomimetic analogs of insect allatostatins. Peptides 2011;32(3):581-586.##Deshmukh R, Purohit HJ. Peptide scaffolds: flexible molecular structures with diverse therapeutic potentials. Int J Pept Res Ther 2012;18(2):125-143.##Heda LC, Sharma R, Pareek C, Chaudhari PB. Synthesis and antimicrobial activity of some derivatives of 5-substituted indole dihydropyrimidines. E-J Chem 2009;6(3):770-774.##Singh UP, Sarma BK, Mishra PK, Ray AB. Antifungal activity of venenatine, an indole alkaloid isolated from Alstonia venenata. Folia Microbiol (Praha) 2000;45(2):173-176.##

Isolation and Culture of Human Spermatogonial Stem cells Derived from Testis Biopsy 2 2 Background: In cancer patients, chemo and radiotherapy can cause infertility by damaging spermatogenesis process. This process is based on self-renewal and differentiation of a rare population of the testicular cells called Spermatogonial Stem Cells (SSCs). Scientists have tried to isolate, enrich and culture Human spermatogonial stem cells, hoping to resolve infertility problems in cancer recovered patients in the future. Methods: Spermatogonial stem cells were isolated and purified from human testicular biopsies sample consisting of at least 500,000 and at most 2,000,000 cells. Two enzymatic digestion steps were performed. Enriching methods, differential plating, and specific culture in serum-free medium with added growth factors: human GDNF, bFGF, EGF and LIF was performed on coated dishes. Results: Human spermatogonial stem cell clusters were observed after 7 to 10 days in specific culture, then after several passages and successful expanding duration of 52 days, the cells were evaluated by three layer immunocytochemistry test (LSAB) to stain GPR125 protein as a surface marker in human spermatogonial stem cells. Conclusion: In current study human spermatogonial stem cell were isolated and expanded with the least manipulations in comparison with the other usual isolation methods like florescent or magnetic activated cell sorting. In contrast to the other SSCs isolation and culture methods, this system is based on the testicular biopsies against large samples, thus suggested method in this study is closer to clinical usage in the future. 54 61 Leila Goharbakhsh Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad UniversityReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad UniversityReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR IranIran Arash Mohazzab Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Sheida Salehkhou Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mahnaz Heidari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Amir-Hassan Zarnani Nanobiotechnology Research Center, Avicenna Research Institute, ACECRImmunology Research Center, Tehran University of Medical Sciences Nanobiotechnology Research Center, Avicenna Research Institute, ACECRImmunology Research Center, Tehran University of Medical Sciences IranIran Kazem Parivar Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University Iran Mohammad Mehdi Akhondi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran CultureEnrichmentGPR125Human spermatogonial stem cellInfertilityIsolation 109.pdf Amirjannati N, Sadeghi M, Hosseini Jadda SH, Ranjbar F, Kamali K, Akhondi MA. Evaluation of semen quality in patients with malignancies referred for sperm banking before cancer treatment. Andrologia 2011;43(5):317-320.##Woodruff TK, Snyder KA. Oncofertility fertility preservation for cancer survivors. New York: Springer Verlag; 2007.##Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972;52(1):198-236##4. Dym M, Kokkinaki M, He Z. Spermatogonial stem cells: mouse and human comparisons. Birth Defects Res C Embryo Today 2009;87(1):27-43.##Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavala S, et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 2007;449(7160):346-350.##Golestaneh N, Kokkinaki M, Pant D, Jiang J, DeStefano D, Fernandez-Bueno C, et al. Pluripotent stem cells derived from adult human testes. Stem Cells Dev 2009;18(8):1115-1126.##Izadyar F, Wong J, Maki C, Pacchiarotti J, Ramos T, Howerton K, Yuen C, et al. Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Hum Reprod 2011;26(6):1296-1306.##He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M. Isolation, characterization, and culture of human spermatogonia. Biol Reprod 2009;82(2):363-372.##Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003;69(2):612-616.##Dirami G, Ravindranath N, Pursel V, Dym M. Effects of stem cells factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol Reprod 1999;61(1):225-230.##Sadri-Ardekani H, Mizrak SC, van Daalen SK, Kor-ver CM, Roepers-Gajadien HL, Koruji M, et al. Propagation of human spermatogonial stem cells in vitro. JAMA 2009;302(19):2127-2134.##Amirjannati N, Heidari-Vala H, Akhondi MA, Hosseini Jadda SH, Kamali K, Sadeghi MR. Comparison of intracytoplasmic sperm injection outcomes between spermatozoa retrieved from testicular biopsy and from ejaculation in cryptozoospermic men. Andrologia 2011;44(Suppl 1):704-709.##Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, et al. Towards a knowledge-based human protein atlas. Nat Biotechnol 2010;28(12):1248-1250.##Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994;91(24):11298-11302.##Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996;2(6):693-696.##Matzuk MM. Germ-line immortality. PNAS 2004;101(47):16395-16396.##Izadyar F, Spierenberg GT, Creemers LB, den Ouden K, de Rooij DG. Isolation and purification of type A spermatogonia from the bovine testis. Reproduction 2002;124(1):85-94.##Folkman J, Haudenschild CC, Zetter BR. Long-term culture of capillary endothelial cells. Proc Natl Acad Sci USA 1979;76(10):5217-5221.##Shinohara T, Avarbock MR, Brinster RL. Beta 1- and alpha 6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA 1999;96(10):5504-5509.##Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69(1):11-25.##Howard GC, Kaser MR. Making and using antibodies: A practical handbook. In: Ramos Vara JA, Saettele JA (eds). Immunohistochemical methods. United Kingdom: Taylor & Francis; 2006, 273-314.##Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 2004;71(3):722-731.##Hofmann MC, Braydich-Stolle L, Dym M. Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 2005;279(1):114-124.##Shinohara T, Brinster RL. Brinster, enrichment and transplantation of spermatogonial stem cells. Int J Androl 2000;23(Suppl 2):89-91.##Miller SR. Assessment of nycodenz gradient on enrichment and culture of perinatal porcine spermatogonial stem cells. [master's thesis]. [NCSU libraries]: NC State University; 2006.155p. ##

A Technique for Facile and Precise Transfer of Mouse Embryos 2 2 Background: Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. Methods: In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. Results: Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software (ver. 9.2). The average live birth rates in the novel methods was significantly higher (42.4%) than the conventional method (21.7%, p<0.05). Conclusion: In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium. 62 65 Ali Sarvari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Naderi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Reza Sadeghi Reproductive Biotechnology Research Center, Avicenna Research Institute Reproductive Biotechnology Research Center, Avicenna Research Institute Iran Mohammad Mehdi Akhondi Reproductive Biotechnology Research Center, Avicenna Research Institute Reproductive Biotechnology Research Center, Avicenna Research Institute Iran Embryo transferMicropipetteMiceEmbryo 110.pdf Van Keuren ML, Saunders TL. Rederivation of transgenic and gene-targeted mice by embryo transfer. Transgenic Res 2004;13(4):363-371. ##Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64(2):382-391.##Kaneda H, Taguma K, Suzuki C, Ozaki A, Nakamura C, Hachisu A, Kobayashi K, et al. An optimal embryo transfer condition for the effective production of DBA/2J mice. Exp Anim 2007;56(5):385-388.##Rose C, Schwegler H, Hanke J, Yilmazer-Hanke DM. Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice. Theriogenology 2012;77(9):1883-1893. ##Tarkowski AK. Experimental studies on regulation in the development of isolated blastomeres of mouse eggs. Acta Theriol 1959;3:191-287.##Whittingham DG. Fertilization of mouse eggs in vitro. Nature 1968;220(5167):592-593.##Zhang Z, Lv X, Wang Y, Chen Y, Zheng R, Sun H, Bian G, et al. Success of murine embryo transfer increased by a modified transfer pipette. J Reprod Dev 2009;55(1):94-97.##Mc Laren A, Michie D. Studies on the transfer of fertilized mouse eggs to uterine foster-mothers. I. Factors affecting the implantation and survival of native and transferred eggs. J Exp Biol 1956;33:397-416.##Rafferty RA. Methods in experimental embryology of the mouse. Baltimore; Johns Hopkins Press: 1970.##Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the mouse embryo. 2nd ed. New York; Cold Spring Harbor Laboratory Press: 1994.##Goto Y, Noda Y, Narimoto K, Umaoka Y, Tokura T, Mori T. Pregnancy achieved by transferring blastocysts into endometrial stroma in mice. Hum Reprod 1992; 7(5):681-684.##Michael AG, Shannon B, Brett TS. Transcervical transfer of mouse embryos using the non-surgical embryo transfer (NSET™) device. BioTechniques 2009;47:919-924. ##Chin HJ, Wang CK. Utero-tubal transfer of mouse embryos. Genesis 2001;30(2):77-81.##Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the mouse embryo: A laboratory manual. 3rd ed. Cold Spring Harbor, New York; Cold Spring Harbor Laboratory Press: 2003, 161–208.##Englert Y, Puissant F, Camus M, Van Hoeck J, Leroy F. Clinical study on embryo transfer after human in vitro fertilization. J In Vitro Fertil Embryo Transfer 1986;3(4):243-246.##Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in vitro fertilization. Fertil Steril 1990;54(4):678-681.##Wood EG, Batzer F.R, Go KJ, Gutmann JN, Corson SL. Ultrasound guided soft catheter embryo transfers will improve pregnancy rates in in-vitro fertilization. Hum Reprod 2000;15(1):107-112.##Visser DS, Fourie FL, Kruger HF. Multiple attempts at embryo transfer: effects on pregnancy outcome in an in vitro fertilization and embryo transfer program. J Assist Reprod Genetics 1993;10(1):37-43.##Tomás C, Tikkinen K, Tuomivaara L, Tapanainen JS, Martikainen H. The degree of difficulty of embryo transfer is an independent variable for predicting pregnancy in in-vitro fertilization treatments. Hum Reprod 2002;17(10):2632-2635.##