BACULOVIRUS-MEDIATED GENETIC MODIFICATION OF HUMAN EMBRYONIC STEM CELLS Du Juan NATIONAL UNIVERSITY OF SINGAPORE 2008 BACULOVIRUS-MEDIATED GENETIC MODIFICATION OF HUMAN EMBRYONIC STEM CELLS DU JUAN (B Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE AND INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY Jan 2008 ACKNOWLEDGMENTS First of all, I would like to express my gratitude to my supervisor, Dr Wang Shu, Associate Professor, Department of Biological Science, National University of Singapore and Group Leader, Institute of Bioengineering and Nanotechnology, for his continuous support and patient supervision during my entire post-graduate study period I would also like to acknowledge our excellent gene delivery group at the Institute of Bioengineering and Nanotechnology for providing a fantastic environment and an exceptional research atmosphere for the study Special acknowledgments go to Dr Zeng Jieming for his patient cooperation and many useful discussions regarding my research project, and to Dr Wu Chunxiao for his helpful suggestions for experiments and critical review of this thesis This thesis is dedicated to my father Du Weijun and my mother Tian Lihua, whose love, encouragement, and support have always been my greatest inspiration I TABLE OF CONTENTS Contents Page ACKNOWLEDGMENTS I TABLE OF CONTENTS II SUMMARY VII LIST OF PUBLICATIONS IX LIST OF TABLES X LIST OF FIGURES .XI ABBREVIATIONS XIII CHAPTER ONE: INTRODUCTION 1.1 General Introduction 1.1.1 Human Embryonic Stem Cells 1.1.2 Non-Viral Genetic Modification Systems for hES Cells 1.1.3 Viral Vectors for Genetic Modification of hES Cells 1.1.4 Baculovirus Vectors Mediated Gene Delivery 1.2 Purpose of This Study 1.3 Specific Objectives CHAPETR TWO: ENGINEERING TRANSGENE EXPRESSION CASSETTES FOR EFFICIENT AND TRANSIENT BACULOVIRAL TRANSDUCTION OF HUMAN EMBRYONIC STEM CELLS 11 2.1 Introduction 12 II 2.1.1 Current Status of Transient Transgene Transfer in hES Cells 12 2.1.2 Promoters for Transgene Expression in Mammalian Cells 13 2.1.3 Viral Genetic Modulators 14 2.1.4 Objectives 15 2.2 Material and Methods 16 2.2.1 Transfer Plasmids and Baculovirus Preparation 16 2.2.2 Maintenance of hES cells and Baculoviral Transduction 19 2.2.3 FACS Analysis 21 2.2.4 Immunocytochemistry 21 2.3 Results 22 2.3.1 Optimization of Baculoviral Transduction Method 22 2.3.2 Baculoviral Vectors Effectively Mediate Gene Transfer to hES Cells 24 2.3.3 Various Promoters Show Differential Efficiency for Baculoviral Transduction of HES-1 27 2.3.4 WPRE, not ITR Sequences, Efficiently Enhanced Baculoviral Transduction of HES-1 30 2.3.5 A Hybrid Promoter Further Improved Baculoviral Transduction of HES-1 33 2.3.6 Transgene Expression Cassettes Optimized in HES-1 Also Displayed Better Efficiency for Baculoviral Transduction of HES-3 and Differentiated HES-1 35 III 2.3.7 Baculoviral transduction of hES cells was transient and had no effect on cell proliferation in vitro 37 2.4 Discussion 39 CHAPTER THREE: HYBRID BACULOVIRUS VECTORS FOR LONG-TERM STABLE TRANSGENE EXPRESSION 45 3.1 Introduction 46 3.1.1 Hybrid Viral Vectors 47 3.1.2 Objectives 48 3.2 Materials and Methods 49 3.2.1 Plasmid Construction and Virus Preparation 49 3.2.2 Cell Line Maintenance and Transduction 51 3.2.3 Maintenance and Differentiation of hES cells 52 3.2.4 Viral Transduction and FACS analysis 54 3.2.5 Genomic DNA isolation and Nested PCR 55 3.2.6 TAIL-PCR 58 3.3 Stable Transgene Expression is Obtained in A Cell Line and in hES Cells: the Single-Vector Approach 62 3.3.1 Hybrid Baculovirus Vectors Mediated Stable Transgene Expression in NT2 Cells 62 3.3.2 Hybrid Baculovirus Vectors Mediated Stable Transgene Expression in hES Cells 64 IV 3.3.3 Transgene Expression is Maintained During hES Cell Differentiation 67 3.3.4 Nested PCR and TAIL PCR Were Employed To Detect Sequences Flanking the Integrated Transgene Cassette 70 3.4 Stable Transgene Expression is obtained in A Cell Line and in hES Cells: A Co-transduction Approach 73 3.4.1 Sustainable transgene expression can be achieved by co-transduction of ITR-and rep-containing baculoviral vectors 73 3.4.2 Nested-PCR Detection of the Transgene Integration Sites on Chr19 in the Genomic DNA of Stably Transduced HeLa Cells 77 3.4.3 Hybrid Baculovirus Vectors Mediated Stable Transgene Expression in hES Cells 81 3.4.4 Analysis of hES Genomic DNA Samples by Nested-PCR, TAIL-PCR and the DNA Walking Speed Up Kit 82 3.5 Discussion 84 CHAPTER FOUR: THE EFFECTS OF BACULOVIRAL TRANSDUCTION ON HUMAN EMBRYONIC STEM CELLS 87 4.1 Introduction and Objective 88 4.2 Materials and Methods 88 4.2.1 Vector Construction and Virus Preparation 88 4.2.2 Maintenance and Differentiation of hES Cells 89 4.2.3 Viral Transduction 91 V 4.2.4 Reverse Transcription-PCR Analysis 91 4.2.5 Immunohistochemistry and Karyotypic Analysis 93 4.2.6 Teratoma Formation 93 4.3 Baculoviral Transduction Has No Effects on hES Cells’ Stem Cell Properties 94 4.3.1 Baculoviral Transduction Did Not Affect hES Cell Growth 94 4.3.2 Baculoviral Transduction Did Not Change hES Phenotype 95 4.3.3 Baculoviral Transduction Did Not Affect hES Cell Pluripotency97 4.4 Discussions 103 CHAPTER FIVE: CONCLUSION 104 5.1 Results and Implications 105 5.1.1 Efficient and Controlled Transient Transgene Expression Can be Achieved by Using Different Promoters and Genetic Modulators105 5.1.2 Hybrid Baculovirus Vectors Allow Stable Transgene Expression 106 5.1.3 Baculoviral Transduction Has No Effects on the Stem Cell Properties of hES Cells 107 References Cited 109 VI SUMMARY The main purpose of the studies in this thesis was to explore and engineer baculoviral vectors for the genetic modification of human embryonic stem cells Although lentiviral vectors are widely used in gene transfer to human embryonic stem cells, there is still a need for vectors and methods to achieve efficient and safe genetic modification of these cells Our studies employed baculovirus vectors to mediate transgene expression, either transiently or stably, as the tools for genetic modification First, we engineered the baculovirus vectors by choosing various promoters and viral modulators to achieve desirable level of transient transgene expression in human embryonic stem cells Efficient gene transfers to human embryonic stem cells were obtained after optimization of the promoters and modulators Second, two elements from adeno-associate virus, ITR sequence and the rep gene were inserted into the baculovirus vectors together with transgene expression cassette We achieved stable transgene expression in human embryonic stem cells for up to moths We also characterized the integration sites of the transgene expression cassette VII Finally, the effects of the baculoviral transduction of the human embryonic stem cells were investigated The transduction of human embryonic stem cells by baculovirus vectors had no effects on the cell growth and pluripotency The formation of teratoma in vivo further confirmed the pluripotency of the transduced cells In summary, the information gained from this research suggest that baculovirus vectors can be engineered to mediate both transient and stable expression in human embryonic stem cells for the purpose of genetic modification, thus facilitating the realization of the great potential of these cells in basic researches and clinical applications VIII 5.1 Results and Implications The main purpose of this study was to explore baculovirus as a gene delivery vector for the genetic modification of hES cells We employed different promoters and viral modulators, to test baculovirus vectors intensively for their ability to mediate efficient but transient transgene expression in hES cells Stable expression was achieved when two elements from AAV were incorporated to produce hybrid baculovirus vectors, both for the single-vector approach and for co-transduction approach As described in the previous chapters, the research work of this thesis can be divided into two parts In this chapter, the main findings of these two parts of study, together with their implications will be discussed 5.1.1 Efficient and Controlled Transient Transgene Expression Can be Achieved by Using Different Promoters and Genetic Modulators As shown in Chapter 2, we have successfully achieved efficient and transient transgene expression in hES cells using baculoviral transduction The transgene expression can be seen at various transduction efficiencies and levels, depending on the choice of promoters and viral modulators A time-course study showed a gradual decline of the transgene expression, which became unobservable days post-transduction Therefore, baculoviral vectors are powerful tools for the genetic modification of hES cells; by engineering the expression cassettes, a spectrum of transient transgene 105 expression levels can be obtained The transient and controllable nature of baculovirus makes it very useful for the delivery of genes involved in cell differentiation to hES cells Baculoviral vectors equipped with appropriate promoters and viral modulators can be applied to the genetic engineering of hES cells to direct their differentiation into different lineages, thus facilitating fundamental developmental studies as well as supplying a source of cells for the regenerative medicine 5.1.2 Hybrid Baculovirus Vectors Allow Stable Transgene Expression As presented in Chapter 3, stable transgene expression in hES cells can be observed By using a hybrid virus approach (i.e., by including a rep gene and ITR sequences from AAV into the baculovirus), the duration of transgene expression in hES cells was extended significantly, so that stably transduced hES cells could be selected and expanded Although lentivirus vectors have been most widely used for the genetic modification of hES cells, these viruses appear to preferentially integrate into chromatin regions characterized by an open structure, a hallmark of actively transcribed genes (Cereseto A et al, 2004) The hybrid vector contains the AAV ITR sequence and the rep gene for targeting and inserting the transgene cassette into a defined region on human chromosome19 q13.3 Our study has for the first time shown that such a hybrid vector can be used for the long-term transgene expression in hES cells The transgene expression was sustained during both cell proliferation and differentiation process However, the 106 integration sites in hES cells still need to be extensively characterized mEFs that can support the growth of hES cells would aid the selection of pure hES cell populations 5.1.3 Baculoviral Transduction Has No Effects on the Stem Cell Properties of hES Cells As demonstrated in Chapter 4, baculoviral transduction did not affect the stem cell properties of hES cells The transduced cells still proliferated normally without morphological changes, and maintained markers for pluripotency as demonstrated by RT-PCR and immunostaining They differentiated into neuronal cells and cardiomyocytes and maintained their normal karyotype The formation of teratomas was also observed All these results demonstrate that baculoviral transduction has no effect on the stemness of hES cells Thus, baculovirus vectors can safely be sued as vectors for the genetic modification of hES cells 5.2 Conclusion The research work presented in this thesis investigated the use of baculovirus as a tool for the genetic modification of human embryonic stem cells, for both the transient and stable transgene expression Moreover, the effects of baculoviral transduction on hES cells were also examined extensively The transduced cells still maintained their stem cells properties, 107 indicating that the baculovirus vectors can be used safely to genetically modify hES cells The results presented in this thesis suggest that baculovirus is a powerful tool for the introduction of transgenes into hES cells Thus, the genetic modification of the hES cells can be achieved Baculovirus vectors can be engineered to realize the great potential of hES cells, advancing the understanding of early human development and providing various types of cells for regenerative medicine 108 References Cited Ben-Dor, I., Itsykson, P., Goldenberg, D., Galun, E., and Reubinoff, B E (2006) Lentiviral Vectors Harboring a Dual-Gene System Allow High and Homogeneous Transgene Expression in Selected Polyclonal Human Embryonic Stem Cells Molecular Therapy 14, 255-267 Boyce, F M., and Bucher, N L R (1996) Baculovirus-mediated gene transfer into mammalian cells (National Acad Sciences), pp 2348-2352 Brooks, A R., Harkins, R N., Wang, P., Qian, H S., Liu, P., and Rubanyi, G M (2004) Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle J Gene Med 6, 395-404 Burns, J C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J (1993) Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors: Concentration to Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells Proceedings of the National Academy of Sciences USA 90, 8033-8037 Carbonell, L F., Klowden, M J., and Miller, L K (1985) Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells Journal of Virology 56, 153-160 Carbonell, L F., and Miller, L K (1987) Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines Applied and Environmental Microbiology 53, 1412-1417 Cereseto, A., and Giacca, M (2004) Integration site selection by retroviruses AIDS Rev 6, 13-21 Chikhlikar, P., de Arruda, L B., Agrawal, S., Byrne, B., Guggino, W., August, J T., and Marques Jr, E T (2004) Inverted terminal repeat sequences of 109 adeno-associated virus enhance the antibody and CD8 responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera Virology 323, 220-232 Costantini, L C., Bakowska, J C., Breakefield, X O., and Isacson, O (2000) Gene therapy in the CNS Gene Ther 7, 93-109 Eiges, R., Schuldiner, M., Drukker, M., Yanuka, O., Itskovitz-Eldor, J., and Benvenisty, N (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells Current Biology 11, 514-518 Gerrard, L., Zhao, D., Clark, A J., and Cui, W (2005) Stably Transfected Human Embryonic Stem Cell Clones Express OCT4-Specific Green Fluorescent Protein and Maintain Self-Renewal and Pluripotency (AlphaMed Press), 124-133 Ghosh, S., Parvez, M K., Banerjee, K., Sarin, S K., and Hasnain, S E (2002) Baculovirus as Mammalian Cell Expression Vector for Gene Therapy: An Emerging Strategy Molecular Therapy 6, 5-11 Groner, A., Granados, R R., and Burand, J P (1984) Interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines Intervirology 21, 203-209 Gropp, M., Itsykson, P., Singer, O., Ben-Hur, T., Reinhartz, E., Galun, E., and Reubinoff, B E (2003) Stable Genetic Modification of Human Embryonic Stem Cells by Lentiviral Vectors Molecular Therapy 7, 281-287 Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C S., Pawliuk, R., and Morillon, E (2003) LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1 Science 302, 415-419 Hartig, P C., Cardon, M C., and Kawanishi, C Y (1992) Effect of baculovirus on selected vertebrate cells Dev Biol Stand 76, 313-317 110 Hay, D C., Sutherland, L., Clark, J., and Burdon, T (2004) Oct-4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast Differentiation Markers in Human and Mouse Embryonic Stem Cells (AlphaMed Press), pp 225-235 Huser, D., and Heilbronn, R (2003) Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency J Gen Virol 84, 133-137 Huser, D., Weger, S., and Heilbronn, R (2002) Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR J Virol 76, 7554-7559 Hyslop, L., Stojkovic, M., Armstrong, L., Walter, T., Stojkovic, P., Przyborski, S., Herbert, M., Murdoch, A., Strachan, T., and Lako, M (2005) Downregulation of NANOG Induces Differentiation of Human Embryonic Stem Cells to Extraembryonic Lineages (AlphaMed Press), pp 1035-1043 Johnston, K M., Jacoby, D., Pechan, P A., Fraefel, C., Borghesani, P., Schuback, D., Dunn, R J., Smith, F I., and Breakefield, X O (1997) HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells Hum Gene Ther 8, 359-370 Kawabata, K., Sakurai, F., Yamaguchi, T., Hayakawa, T., and Mizuguchi, H (2005) Efficient gene transfer into mouse embryonic stem cells with adenovirus vectors Mol Ther 12, 547-554 Kim, S., Kim, G J., Miyoshi, H., Moon, S H., Ahn, S E., Lee, J H., Lee, H J., Cha, K Y., and Chung, H M (2007) Efficiency of the elongation factor-1alpha promoter in Mammalian embryonic stem cells using lentiviral gene delivery systems Stem Cells Dev 16, 537-546 111 Klein, R L., Hamby, M E., Gong, Y., Hirko, A C., Wang, S., Hughes, J A., King, M A., and Meyer, E M (2002) Dose and Promoter Effects of Adeno-Associated Viral Vector for Green Fluorescent Protein Expression in the Rat Brain Experimental Neurology 176, 66-74 Kobayashi, M., Tanaka, A., Hayashi, Y., and Shimamura, S (1997) The CMV enhancer stimulates expression of foreign genes from the human EF-1 alpha promoter Anal Biochem 247, 179-181 Kobayashi, N., Rivas-Carrillo, J D., Soto-Gutierrez, A., Fukazawa, T., Chen, Y., Navarro-Alvarez, N., and Tanaka, N (2005) Gene delivery to embryonic stem cells Birth Defects Res C Embryo Today 75, 10-18 Kost, T A., and Condreay, J P (2002) Recombinant baculoviruses as mammalian cell gene-delivery vectors Trends Biotechnol 20, 173-180 Kost, T A., Condreay, J P., and Jarvis, D L (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells Nat Biotechnol 23, 567-575 Kotin, R M., Linden, R M., and Berns, K I (1992) Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination Embo J 11, 5071-5078 Kumar, M., Bradow, B P., and Zimmerberg, J (2003) Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64 Hum Gene Ther 14, 67-77 Kyba, M., Perlingeiro, R C., and Daley, G Q (2002) HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors Cell 109, 29-37 Kyba, M., Perlingeiro, R C., Hoover, R R., Lu, C W., Pierce, J., and Daley, G Q (2003) Enhanced hematopoietic differentiation of embryonic stem cells 112 conditionally expressing Stat5 Proc Natl Acad Sci U S A 100 Suppl 1, 11904-11910 Lakshmipathy, U., Pelacho, B., Sudo, K., Linehan, J L., Coucouvanis, E., Kaufman, D S., and Verfaillie, C M (2004) Efficient Transfection of Embryonic and Adult Stem Cells (AlphaMed Press), pp 531-543 Lam, P., Hui, K M., Wang, Y., Allen, P D., Louis, D N., Yuan, C J., and Breakefield, X O (2002) Dynamics of transgene expression in human glioblastoma cells mediated by herpes simplex virus/adeno-associated virus amplicon vectors Hum Gene Ther 13, 2147-2159 Liew, C G., Draper, J S., Walsh, J., Moore, H., and Andrews, P W (2007) Transient and stable transgene expression in human embryonic stem cells Stem Cells 25, 1521-1528 Liu, B H., Wang, X., Ma, Y X., and Wang, S (2004) CMV enhancer/human PDGF-beta promoter for neuron-specific transgene expression Gene Ther 11, 52-60 Liu, Q., Perez, C F., and Wang, Y (2006) Efficient Site-Specific Integration of Large Transgenes by an Enhanced Herpes Simplex Virus/Adeno-Associated Virus Hybrid Amplicon Vector Journal of Virology 80, 1672-1679 Loeb, J E., Cordier, W S., Harris, M E., Weitzman, M D., and Hope, T J (1999) Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy Hum Gene Ther 10, 2295-2305 Ma, Y., Ramezani, A., Lewis, R., Hawley, R G., and Thomson, J A (2003) High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors (AlphaMed Press), pp 111-117 113 McCarty, D M., Young, S M., Jr., and Samulski, R J (2004) Integration of adeno-associated virus (AAV) and recombinant AAV vectors Annu Rev Genet 38, 819-845 Med, J G (2004) Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle J Gene Med 6, 395-404 Mossman, A K., Sourris, K., Ng, E., Stanley, E G., and Elefanty, A G (2005) Mixl1 and Oct4 Proteins Are Transiently Co-Expressed in Differentiating Mouse and Human Embryonic Stem Cells Stem Cells Dev 14, 656-663 Mountford, P., Nichols, J., Zevnik, B., O'Brien, C., and Smith, A (1998) Maintenance of pluripotential embryonic stem cells by stem cell selection Reprod Fertil Dev 10, 527-533 Nightingale, S J., Hollis, R P., Pepper, K A., Petersen, D., Yu, X J., Yang, C., Bahner, I., and Kohn, D B (2006) Transient gene expression by nonintegrating lentiviral vectors Mol Ther 13, 1121-1132 Niwa, H., Yamamura, K., and Miyazaki, J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector Gene 108, 193-199 Palombo, F., Monciotti, A., Recchia, A., Cortese, R., Ciliberto, G., and La Monica, N (1998) Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus-Adeno-Associated Virus Vector Journal of Virology 72, 5025-5034 Paterna, J C., and Bueler, H (2002) Recombinant adeno-associated virus vector design and gene expression in the mammalian brain Methods 28, 208-218 Pfeifer, A., Ikawa, M., Dayn, Y., and Verma, I M (2002) Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem 114 cells and preimplantation embryos Proceedings of the National Academy of Sciences USA 99, 2140-2145 Philip, R., Brunette, E., Kilinski, L., Murugesh, D., McNally, M A., Ucar, K., Rosenblatt, J., Okarma, T B., and Lebkowski, J S (1994) Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes Mol Cell Biol 14, 2411-2418 Philippe, S., Sarkis, C., Barkats, M., Mammeri, H., Ladroue, C., Petit, C., Mallet, J., and Serguera, C (2006) Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo Proc Natl Acad Sci U S A 103, 17684-17689 Pieroni, L., and La Monica, N (2001) Towards the use of baculovirus as a gene therapy vector Curr Opin Mol Ther 3, 464-467 Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., and Goldstein, R S (2005) Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells Stem Cells 23, 923-930 Reubinoff, B E., Itsykson, P., Turetsky, T., Pera, M F., Reinhartz, E., Itzik, A., and Ben-Hur, T (2001) Neural progenitors from human embryonic stem cells Nat Biotechnol 19, 1134-1140 Reubinoff, B E., Pera, M F., Fong, C Y., Trounson, A., and Bongso, A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro Nat Biotechnol 18, 399-404 Saenz, D T., Loewen, N., Peretz, M., Whitwam, T., Barraza, R., Howell, K G., Holmes, J M., Good, M., and Poeschla, E M (2004) Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants J Virol 78, 2906-2920 115 Sarkis, C., Serguera, C., Petres, S., Buchet, D., Ridet, J L., Edelman, L., and Mallet, J (2000) Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector Proceedings of the National Academy of Sciences USA 97, 14638-14643 Sawicki, J A., Morris, R J., Monks, B., Sakai, K., and Miyazaki, J (1998) A composite CMV-IE enhancer/beta-actin promoter is ubiquitously expressed in mouse cutaneous epithelium Exp Cell Res 244, 367-369 Schauber, C A., Tuerk, M J., Pacheco, C D., Escarpe, P A., and Veres, G (2004) Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro Gene Ther 11, 266-275 Schroder, A R., Shinn, P., Chen, H., Berry, C., Ecker, J R., and Bushman, F (2002) HIV-1 integration in the human genome favors active genes and local hotspots Cell 110, 521-529 Siemen, H., Nix, M., Endl, E., Koch, P., Itskovitz-Eldor, J., and Brustle, O (2005) Nucleofection of human embryonic stem cells Stem Cells Dev 14, 378-383 Smith-Arica, J R., Thomson, A J., Ansell, R., Chiorini, J., Davidson, B., and McWhir, J (2003) Infection Efficiency of Human and Mouse Embryonic Stem Cells Using Adenoviral and Adeno-Associated Viral Vectors Cloning and Stem Cells 5, 51-62 Strulovici, Y., Leopold, P L., O'Connor, T P., Pergolizzi, R G., and Crystal, R G (2007) Human embryonic stem cells and gene therapy Mol Ther 15, 850-866 Thomson, J A., Kalishman, J., Golos, T G., Durning, M., Harris, C P., Becker, R A., and Hearn, J P (1995) Isolation of a Primate Embryonic Stem Cell 116 Line Proceedings of the National Academy of Sciences USA 92, 7844-7848 Tsunoda, H., Hayakawa, T., Sakuragawa, N., and Koyama, H (2000) Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells Virology 268, 391-401 Vargas, J., Jr., Gusella, G L., Najfeld, V., Klotman, M E., and Cara, A (2004) Novel integrase-defective lentiviral episomal vectors for gene transfer Hum Gene Ther 15, 361-372 Vieweg, J., Boczkowski, D., Roberson, K M., Edwards, D W., Philip, M., Philip, R., Rudoll, T., Smith, C., Robertson, C., and Gilboa, E (1995) Efficient gene transfer with adeno-associated virus-based plasmids complexed to cationic liposomes for gene therapy of human prostate cancer Cancer Res 55, 2366-2372 Volkman, L E., and Goldsmith, P A (1983) In Vitro Survey of Autographa californica Nuclear Polyhedrosis Virus Interaction with Nontarget Vertebrate Host Cells Applied and Environmental Microbiology 45, 1085-1093 Wang, C Y., Guo, H Y., Lim, T M., Ng, Y K., Neo, H P., Hwang, P Y., Yee, W C., and Wang, S (2005) Improved neuronal transgene expression from an AAV-2 vector with a hybrid CMV enhancer/PDGF-beta promoter J Gene Med 7, 945-955 Wang, C Y., Li, F., Yang, Y., Guo, H Y., Wu, C X., and Wang, S (2006) Recombinant baculovirus containing the diphtheria toxin A gene for malignant glioma therapy Cancer Res 66, 5798-5806 Wu, X., Li, Y., Crise, B., and Burgess, S M (2003) Transcription start regions in the human genome are favored targets for MLV integration Science 300, 1749-1751 117 Xia, X., Zhang, Y., Zieth, C R., and Zhang, S C (2007) Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner Stem Cells Dev 16, 167-176 Xin, K Q., Ooki, T., Jounai, N., Mizukami, H., Hamajima, K., Kojima, Y., Ohba, K., Toda, Y., Hirai, S., Klinman, D M., et al (2003) A DNA vaccine containing inverted terminal repeats from adeno-associated virus increases immunity to HIV J Gene Med 5, 438-445 Xiong, C., Tang, D Q., Xie, C Q., Zhang, L., Xu, K F., Thompson, W E., Chou, W., Gibbons, G H., Chang, L J., Yang, L J., and Chen, Y E (2005) Genetic engineering of human embryonic stem cells with lentiviral vectors Stem Cells Dev 14, 367-377 Xu, C., Rosler, E., Jiang, J., Lebkowski, J S., Gold, J D., O'Sullivan, C., Delavan-Boorsma, K., Mok, M., Bronstein, A., and Carpenter, M K (2005) Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium Stem Cells 23, 315-323 Xu, R., Janson, C G., Mastakov, M., Lawlor, P., Young, D., Mouravlev, A., Fitzsimons, H., Choi, K L., Ma, H., Dragunow, M., et al (2001) Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes Gene Ther 8, 1323-1332 Yanez-Munoz, R J., Balaggan, K S., MacNeil, A., Howe, S J., Schmidt, M., Smith, A J., Buch, P., MacLaren, R E., Anderson, P N., Barker, S E., et al (2006) Effective gene therapy with nonintegrating lentiviral vectors Nat Med 12, 348-353 Yang, C C., Xiao, X., Zhu, X., Ansardi, D C., Epstein, N D., Frey, M R., Matera, A G., and Samulski, R J (1997) Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro J Virol 71, 9231-9247 118 Yates, F., and Daley, G Q (2006) Progress and prospects: gene transfer into embryonic stem cells Gene Ther 13, 1431-1439 Yew, N S., Przybylska, M., Ziegler, R J., Liu, D., and Cheng, S H (2001) High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter Mol Ther 4, 75-82 Ying, Q L., Nichols, J., Chambers, I., and Smith, A (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3 Cell 115, 281-292 Zeng, J., Du, J., Zhao, Y., Palanisamy, N., and Wang, S (2007) Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells Stem Cells 25, 1055-1061 Zufferey, R., Donello, J E., Trono, D., and Hope, T J (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors J Virol 73, 2886-2892 Zwaka, T P., and Thomson, J A (2003) Homologous recombination in human embryonic stem cells Nat Biotechnol 21, 319-321 119 ... 1.1.1 Human Embryonic Stem Cells 1.1.2 Non-Viral Genetic Modification Systems for hES Cells 1.1.3 Viral Vectors for Genetic Modification of hES Cells 1.1.4 Baculovirus Vectors Mediated. . .BACULOVIRUS- MEDIATED GENETIC MODIFICATION OF HUMAN EMBRYONIC STEM CELLS DU JUAN (B Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES... sites of the transgene expression cassette VII Finally, the effects of the baculoviral transduction of the human embryonic stem cells were investigated The transduction of human embryonic stem cells