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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 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