Baculovirus mediated genetic modification of human embryonic stem cells

8 CHAPETR TWO: ENGINEERING TRANSGENE EXPRESSION CASSETTES FOR EFFICIENT AND TRANSIENT BACULOVIRAL TRANSDUCTION OF HUMAN EMBRYONIC STEM CELLS ..... Although lentiviral vectors are widely

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

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

1.1 General Introduction 2

1.1.1 Human Embryonic Stem Cells 2

1.1.2 Non-Viral Genetic Modification Systems for hES Cells 3

1.1.3 Viral Vectors for Genetic Modification of hES Cells 4

1.1.4 Baculovirus Vectors Mediated Gene Delivery 5

1.2 Purpose of This Study 7

1.3 Specific Objectives 8

CHAPETR TWO: ENGINEERING TRANSGENE EXPRESSION CASSETTES FOR EFFICIENT AND TRANSIENT BACULOVIRAL TRANSDUCTION OF HUMAN EMBRYONIC STEM CELLS 11

2.1 Introduction 12

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

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

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

4.2.4 Reverse Transcription-PCR Analysis 914.2.5 Immunohistochemistry and Karyotypic Analysis 934.2.6 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 944.3.2 Baculoviral Transduction Did Not Change hES Phenotype 954.3.3 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 Modulators1055.1.2 Hybrid Baculovirus Vectors Allow Stable Transgene

Expression 1065.1.3 Baculoviral Transduction Has No Effects on the Stem Cell

Properties of hES Cells 107

References Cited 109

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 5 moths We also characterized the integration sites of the transgene expression cassette

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

LIST OF PUBLICATIONS

Publications:

1 Du J, Zeng J, Zhao Y, Wang S The combined use of viral transcriptional

and post-transcriptional regulatory elements to improve baculovirus-mediated transient gene expression in human embryonic stem cells Manuscript in preparation

2 Zeng J, Du J, Zhao Y, Palanisamy N, Wang S Baculoviral

vector-mediated transient and stable transgene expression in human

embryonic stem cells Stem Cells 2007 Apr; 25(4):1055-61

3 Ong ST, Li F, Du J, Tan YW, Wang S Hybrid cytomegalovirus

enhancer-h1 promoter-based plasmid and baculovirus vectors mediate

effective RNA interference Hum Gene Ther 2005 Dec; 16(12):1404-12

The studies presented in this thesis are based on the research work in the above publications and manuscript

LIST OF TABLES

Table 2.1 Expression cassettes determine transgene expression in hES cells

transduced with baculoviral vectors………28

Table 3.1 Nested-PCR primers used to detect of integration sites on chromosome 19……… ……57

Table 3.2 Primers for TAIL-PCR……… 59

Table 3.3 TAIL-PCR programs……….60

Table 4.1 Primers for RT-PCR analysis……… 92

Figure2.5 Flow cytometric analysis of transgene expression in HES-1 cells

mediated by baculoviral vectors carrying different expression cassette (MOI

100) ………32

Figure2.6 Flow cytometric analysis of transgene expression in HES-1 cells

mediated by baculoviral vectors carrying various expression cassette (MOI

10) ………34

Figure2.7 Flow cytometric analysis of transgene expression in HES-3 cells

mediated by baculoviral vectors carrying various expression

Figure 3.3 Maintenance of transgene expression after neural differentiation

of human embryonic stem (hES) cells stably transduced with a hybrid baculovirus harboring a cytomegalovirus promoter-driven enhanced green

fluorescent protein (eGFP) gene………69

Figure 3.4 aavs1 primer binding sites on chr19………71

Figure 3.5 Flow cytometric analysis of the EGFP+ population of HeLa cells co-transduced by ITR and Rep-containing baculoviral vectors………75

Figure 3.6 Detection of site-specific integration mediated by hybrid baculovirus vectors in HeLa cells by nested-PCR………78

Figure 3.7 Transgene and aavs1 junction sequence from HeLa cells clone………80

Figure 3.8 Stable expressions of a reporter gene in hES cells can be achieved by co-transduction of hybrid baculoviral vectors………83

Figure 4.1 Effects of baculovirus infection using transient expression vectors

on the expression of molecular markers for hES cells and the three germ

layers in embryoid bodies………96

Figure 4.2 RT-PCR analysis of markers for the three germ layers of baculovirus-transduced embryoid bodies……….98

Figure 4.3 Effects of baculovirus infection using stable expression vectors on the expression of molecular markers and the karyotype of stably transduced hES

Figure 4.4 Immunostaining and teratoma formation of the baculoviral transduced hES cells………102

ABBREVIATIONS

AAV adeno-associate virus

AcMNPV Autographa californica multiple

CNS central nervous system

DMEM Dulbecco’s modified eagle’s medium

EF1α elongation factor 1α

eGFP enhanced green fluorescent protein

ES cells embryonic stem cells

FBS fetal bovine serum

hES cells human embryonic stem cells

HIV human immunodeficiency virus

HSV herpes simplex virus

ITR inverted terminal repeat

MOI multiplicity of infection

MCS multiple cloning site

CHAPTER ONE

INTRODUCTION

1.1 General Introduction

Embryonic stem (ES) cells as a renewable cell source for regenerative medicine, for pharmaceutical research and development and for developmental biology studies, are currently the subject of intensive research.Genetic manipulation of these cells with an effective gene delivery strategy is

of great importance in making use of the remarkable potential of ES cells.Possible benefits of genetic manipulation include controlling differentiation of

ES cells, screening and acquiring pure populations of specific types of ES cell-derived cells, altering antigenicity of cells to overcome immune rejection

in transplantation medicine, and providing cell sources with new functional properties to combat specific diseases in the course of ex vivo gene therapy

1.1.1 Human Embryonic Stem Cells

Human embryonic stem (hES) cells are cells derived from the inner cell mass of human blastocysts The hES cell research began with the derivation

of hES cell line and its successful culture in the laboratory (Thomson et al., 1995) Human embryonic stem cells possess two characteristic properties, self-renewal and differentiation capability Self-renewal allows hES cells to be kept in culture and passaged long-term under defined culture conditions without loss of their pluripotency This property of self-renewal will supply research and clinical applications with an unlimited cell source The other interesting feature of hES cells is their differentiation potentials hES cells are

able to differentiate into all the cell types in the body, that is, the cells are pluripotent These two properties make human embryonic stem cells promising and attractive for use in regenerative medicine and studies of human development as well as for pharmaceutical screening

1.1.2 Non-Viral Genetic Modification Systems for hES Cells

In order to realize the great potential of hES cells, efficient and safe methods need to be developed to deliver genes or proteins into these cells The delivery of genes or proteins or certain factors to give stem cells new genetic characteristics is called genetic modification The genetic modification of hES cells can be helpful in the differentiation and isolation of lineage-committed cells, in tracking the process of differentiation, in gene function analysis, and in altering immunogenecity To achieve successful genetic modification, safe and efficient gene delivery systems are crucial Non-viral and viral systems have been developed and optimized for use in genetic manipulation

Non-viral delivery systems, using naked DNA or synthetic oligonucleotides directly, or genes or proteins encapsulated with lipids, peptides or polymers, are widely favored because of their safety Low toxicity

is the most attractive advantage of the non-viral delivery systems However, they can only achieve a certain level of efficiency of transgene or protein delivery, while viral vectors are well known for their high delivery efficiency

Chemically based gene transfection and antibiotic selection have been performed in order to establish stable human ES cell lines (Eiges et al., 2001), but the method’s gene transfer efficiency and the efficiency of subsequent homologous recombination in human ES cells are low Electroporation, a method of choice for introducing foreign DNA into mouse ES cells, can be adapted for the gene transfection of human ES cells (Zwaka and Thomson, 2003) With nucleofection, an electroporation-based method using specific transfection solution and electrical parameters to deliver plasmid DNA into the cell nucleus, a transfection rate of 20% could be achieved in human ES cells (Lakshmipathy et al., 2004) However, electroporation protocols that give satisfactory transfection efficiency are usually detrimental to cells and hES cells do not survive the procedure well (Eiges et al., 2001)

1.1.3 Viral Vectors for Genetic Modification of hES Cells

Modified viruses are also used to deliver transgenes both in vitro and in vivo, mainly due to their high transduction efficiency Various viruses have been investigated and modified for use as delivery vectors, including retroviruses, adenoviruses (Ads), adeno-associated viruses (AAVs), and herpes simplex viruses (HSVs)

Among these commonly used viral vectors, HIV-based lentiviral vectors were the first and most widely used viral vectors for the genetic modification

of hES cells because of their high gene delivery efficiency and their ability to

mediate stable transgene expression during prolonged undifferentiated proliferation in vitro for an observation period of at least 38 weeks (Eiges et al., 2001; Gropp et al., 2003; Ma et al., 2003) Besides lentivirus vectors, adenoviral and adeno-associated viral (AAV) vectors are also able to infect human ES cells (Smith-Arica et al., 2003) Although the potential application

of these viral vectors is enormous, concerns still remain over their immunogenecity in medical use, especially with regard to recombination with endogenous viruses, oncogenic chromosome insertion associated with random integration, non-selective cytotoxicity and pre-existing immune responses against the viruses Furthermore, vector size for gene cloning may

be another limiting factor for certain applications that require large transgene cassettes Thus, there is still a great need for efficient and safe vectors for the genetic modification of hES cells

1.1.4 Baculovirus Vectors Mediated Gene Delivery

In addition to the commonly used viral vectors mentioned above, the

insect cells baculovirus Autographa californica multiple nucleopolyhedrovirus

(AcMNPV)-based vectors are relatively new and have recently emerged as a promising gene delivery vector capable of efficiently transferring genes of interest to diverse mammalian cell types (Ghosh et al., 2002; Kost and Condreay, 2002) The ability of baculovirus to enter certain mammalian cell lines was firstly reported by Volkman and Goldsmith in 1983, while no evidence of viral gene expression was observed in their study Later studies

showed that recombinant viruses containing a promoter that is active in mammalian cells could be used to transduce such cells, and could therefore potentially be used as gene delivery vectors for mammalian cells Carbonell and Miller (1987), using baculovirus with a mammalian gene expression cassette, detected reporter enzyme activity in a human lung carcinoma cell line Baculoviruses with CMV promoter were shown by Condreay and colleagues to infect a broad spectrum of cell lines (Condreay et al., 1999) Recently, baculovirus has also been successfully used as vector to deliver genes into the CNS (Sarkis et al., 2000)

As one of the most promising gene delivery vectors, baculovirus has a number of advantages First, baculovirus has a good biosafety profile The investigation of the interaction between baculovirus and mammalian cells has indicated that baculovirus can enter but cannot replicate in mammalian cells (Carbonell et al., 1985; Carbonell and Miller, 1987; Groner et al., 1984; Hartig

et al., 1992) Thus, no particular attention needs to be paid to the risks associated with replication-competent virus, a major problem in the use of adenovirus vectors as in vivo gene delivery vectors However, further studies will be required to determine whether any of the viral genes are expressed in mammalian cell (Boyce and Bucher, 1996) Second, baculovirus can accommodate large insertions (>20 kb) The rod-shaped AcMNPV has a double-stranded, circular DNA about 130 kb in size and the nucleocapsid structure can accommodate up to 100 kb of foreign DNA This enables the

delivery of large functional gene or of multiple genes simultaneously Third, baculovirus shows broad cell-type tropism More than 40 commonly used cell lines, including some primary cultures, have been reported to be successfully transduced (Ghosh et al., 2002; Kost and Condreay, 2002) Moreover, baculovirus is non-cytotoxic even it was used at high multiplicity of infection (MOI) Fourth, baculovirus is simple to manipulate and produce The AcMNPV vectors are helper virus independent; thus, it is relatively easy to construct baculovirus vector with various transgene cassette and obtain large quantities of high-titer virus stock

Although lentivirus vectors are the most successfully and widely used vectors for the genetic modification of human embryonic stem cells, the random nature of their chromosomal integration has raised safety concerns and the cloning capacity limitation of lentivirus vectors limits the size of the transgene that can be delivered Therefore, new vectors that can mediate efficient and safe gene transfer to hES cells are needed Based on the advantages of baculovirus vectors as described above, in this study, we undertook this study to investigate the suitability of baculovirus as a viral vector for the genetic modification of hES cells

1.2 Purpose of This Study

This thesis will focus on the exploration of baculovirus as a vector for the genetic modification of hES cells, both for transient transgene expression and for long-term transgene expression

First, we constructed baculoviral vectors containing various promoters and viral modulators to achieve different transduction efficiencies and transgene expression intensities in hES cells Second, we constructed baculovirus vectors to which we added two AAV elements to mediate stable, prolonged transgene expressions and investigated chromosome integration sites to address the genotoxicity of these hybrid viral vectors Third, the effects of baculoviral vectors were examined using various approaches in vitro and in vivo to test the effects of this viral vector on the proliferation and differentiation of hES cells

1.3 Specific Objectives

The main investigation of this research can be organized into three parts:

1 Baculovirus was firstly demonstrated to mediate transgene expression

in human embryonic stem cells and baculovirus vectors were then genetically engineered with choice of promoters and viral modulators to achieve various delivery efficiencies and different level of transgene expression as vectors for transient transgene expression

2 Baculoviral vectors that incorporated the two AAV elements were constructed to become hybrid viral vector and stable transgene

expression by these hybrid vectors were observed for a prolonged period and characterized

3 The effects of baculoviral transduction on hES cells were investigated

to determine whether the baculovirus could be used as safe viral vector for the genetic modification of ES cell In vitro and in vivo assays were used to test the effects of baculoviral transduction on the self-renewal and differentiation properties of ES cells

This study demonstrated that baculovirus is an efficient and safe vector for the genetic modification of hES cells both for transient transgene expression and for stable transgene expression, thus making baculovirus an alternative viral vector (if not better than lentiviral vectors), for the genetic modification of ES cells By incorporating different promoters and viral modulators or transgenes, baculoviruses can be used to achieve gene transfer into hES cell thus facilitating stem cell research In addition, baculovirus can be employed to deliver factors or proteins that may direct the differentiation of hES cells to obtain specific lineages Moreover, baculovirus could be used to deliver functional or therapeutic genes into human embryonic stem cells and their derivatives The genetically modified hES cells and derivatives could be probably be applied in cell-based therapy to restore lost functions, diminish abnormal cell population or repair damaged tissues

In the next chapter, we explore the use of baculovirus vectors as genetic modification tools for mediating transient transgene expression

CHAPTER TWO ENGINEERING TRANSGENE EXPRESSION CASSETTES FOR EFFICIENT AND TRANSIENT BACULOVIRAL TRANSDUCTION OF HUMAN EMBRYONIC STEM CELLS

2.1 Introduction

hES cells hold great potential as a system for the study of lineage specification and as an unlimited source of cells for the regenerative medicine To meet these needs, direction differentiation of hES cells is crucial, which involves the upregulation and downregulation of certain differentiation-related genes within a very short time period (Hay et al., 2004; Hyslop et al., 2005; Kawabata et al., 2005; Mossman et al., 2005; Smith-Arica et al., 2003) Thus, a gene delivery vector which can mediate transgene expression transiently and efficiently is of great importance for directing differentiation and lineage commitment (Eiges et al., 2001;

Lakshmipathy et al., 2004; Liew et al., 2007; Siemen et al., 2005)

2.1.1 Current Status of Transient Transgene Transfer in hES Cells

Although various non-viral and viral gene delivery methods have been developed for the genetic modification of hES cells, none of the existing vectors have been proved to be efficient and useful for transient transgene expression in these cells Non-viral methods offer transient expression of the transgene, but the low efficiency and the cytotoxicity of the methods are problematic Viral vectors derived from adenovirus and AAV have shown low efficiency in hES cells transduction (Smith-Arica et al., 2003) Lentiviral vectors are still the most successful and efficient vectors which are used for the genetic modification of hES cells However, these vectors mediate

persistent transgene expression by random integration into chromosomes The random nature of the integration raises biosafety concerns, due to the possibility of insertional mutagenesis (Hacein-Bey-Abina et al., 2003; Schroder et al., 2002; Wu et al., 2003) and reduces the likelihood of lentiviral

vectors being used for efficient transient transgene expression

2.1.2 Promoters for Transgene Expression in Mammalian Cells

The choice of promoter has always been an important issue for gene delivery, as it greatly affects the quantitative expression of the transgene as well as the slicing of the transcripts Promoters such as CAG, PGK, EF1α, U3, CMV, TK and Oct4 have been used in a limited list of viral or non-viral vectors for the genetic modification of hES cells (Strulovici et al., 2007)

Oct4 is a transcription factor with a central regulatory role in controlling of pluripotency of embryonic stem cells and is widely used as a hES cell-specific marker (Mountford et al., 1998; Ying et al., 2003) Comparatively little work has been done on the use of the human Oct4 promoter to drive transgene expression in hES cells One study has demonstrated the activity

of an exogenous Oct4 promoter in driving the expression of eGFP gene in stable hES cell clones (Gerrard et al., 2005)

The CMV promoter is a strong viral promoter widely used for transgene expression in many cell types It has been commonly used to drive the

transgene expression in hES cells, mostly in non-viral vectors to yield low or moderate levels of transgene expression (Yael, Philip et al., 2007).

The promoter of the house-keeping gene EF1α is a popular choice for genetic manipulation of hES cells (Strulovici et al., 2007) In a recently published study, lentiviral vectors containing the EF1α promoter provided gene expression in 14.74% of hES cells, higher than the efficiency of 3.69% provided by a lentiviral vector equipped with the CMV promoter (Kim et al., 2007)

A hybrid promoter approach can be applied to improve promoter strength, resulting in high level of transgene expression in mammalian cells The SV40 enhancer and CMV enhancer, strong viral enhancers of small size, are both commonly fused with promoters of low transcriptional activity to enhance their strength (Niwa et al., 1991; Sawicki et al., 1998) The inclusion of the CMV enhancer upstream of the EF1α promoter has proven to be 4-9 fold more efficient in activity than a construct lacking the CMV enhancer (Kobayashi et al., 1997)

2.1.3 Viral Genetic Modulators

Besides promoters, other DNA elements are also used to regulate transgene expression in mammalian cells The AAV 145bp inverted terminal repeats (ITR) sequence was placed flanking the transgene cassette, the cassette demonstrated higher transgene expression efficiency Although the

mechanism is not clear but the effects of the ITRs were attributed to mRNA stabilization Researchers have reported that transgene expression efficiency in mammalian cells is improved by the use of ITR sequences to flank the transgene expression cassette in viral vectors or plasmids (Chikhlikar et al., 2004; Costantini et al., 2000; Johnston et al., 1997; Lam et al., 2002; Philip et al., 1994; Vieweg et al., 1995; Xin et al., 2003)

Insertion of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) into lentiviral vectors with the EF1α promoter has been reported to increase transgene expression in hES cells (Gropp et al., 2003; Pfeifer et al., 2002) When AAV vectors were used to transduce rat neurons, incorporation of WPRE enhanced transgene expression from the CMV promoter, the neuron-specific enolase promoter, and the CAG promoter (Klein et al., 2002; Loeb et al., 1999; Xu et al., 2001) These findings suggest that the beneficial effect of the viral WPRE element on gene expression is probably promoter-independent Interestingly, the use of WPRE in the above

animal studies also prolonged gene expression

2.1.4 Objectives

The aim of this study was to develop a baculoviral vector for efficient and transient transgene expression in hES cells The activities of various promoters were compared and the delivery efficiency of baculovirus vectors with a combination of viral genetic modulators was also investigated The

optimized vectors developed in this study can be used to deliver transcription factors or regulatory proteins in order to facilitate or direct the differentiation

of hES cells

2.2 Material and Methods

2.2.1 Transfer Plasmids and Baculovirus Preparation

The transfer plasmid pFastBac1 (pFB, Invitrogen, Carlsbad, CA, http://www.invitrogen.com) was used to accommodate the eight different expression cassettes studied (Table 2.1) and to generate recombinant baculoviruses To construct the transfer plasmid pFB-CMV.eGFP, the human cytomegalovirus (CMV) immediate early gene promoter and enhancer (CMV promoter) and enhanced green fluorescent protein (eGFP) gene from pEGFP-C1 (Clontech, Mountain View, CA, http://www.clontech.com) were inserted between the BamHI and EcoRI sites of pFastBac1 To construct the transfer plasmid pFB-Oct4.eGFP containing the Oct4 promoter, the human Oct4 promoter fragment was cut from pOct4-PGL-3 basic (kindly provided by Professor NG Huck Hui, Genome Institute of Singapore, Singapore) with BglII and Agel and inserted between the BamHI and AgeI sites of pFB-CMV.eGFP to replace the CMV promoter The transfer plasmid pFB-EF1α.eGFP containing the human elongation factor-1α promoter (EF1α promoter) was cloned by inserting a human elongation factor-1α (EF1α) promoter from pEF1V5-HisA (Invitrogen) between the BamHI and EcoRI

sites of pFastBac1 Next, the enhanced green fluorescent protein (eGFP) gene from pEGFP-C1 was amplified using the forward primer 5’-CAAGGAATTCCGCCACCATGGTGAGCAA-3’ and the reverse primer 5’-ATTACTAGTCTACTTGTACAGCTCGTC-3’ by the polymerase chain reaction (PCR) and inserted between the EcoRI and SpeI sites

The transfer plasmid pFB-EF1α.eGFP-WPRE that incorporates the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was constructed by replacing the eGFP gene in pFB-EF1α.eGFP with the eGFP-WPRE fragment from psubCMV-eGFP-WPRE (kindly provided by Professor H Büeler, University of Zurich, Switzerland) To produce pFB-EF1α.eGFP+ITRs and pFB-EF1α.eGFP-WPRE+ITRs, which contain expression cassettes flanked by AAV ITR sequences, the EF1α promoter from pEF1V5-HisA (Invitrogen) was inserted between the KpnI and HindIII sites of a previously described plasmid pAAV-MCS-luc (Wang et al., 2005) and the eGFP gene or the eGFP-WPRE fragment was used to replace the luc gene between the HindIII and XbaI sites To construct the transfer plasmids pFB-eCMV.EF1α.eGFP and pFB-eCMV.pEF1α.eGFP-WPRE, which include the hybid promoter, the CMV immediate early gene enhancer fragment was PCR-amplified from pEGFP-C1 and inserted into pFB-EF1α.eGFP and pFB-EF1α.eGFP-WPRE upstream of the EF1α promoter at the BamHI site (Figure 2.1)

pA eGFP

pCMV

pA eGFP

pA eGFP

pOct-4

pA eGFP

pA eGFP

pA eGFP

L-ITR

R-ITR L-ITR

pA eGFP

eCMV

pA eGFP

eCMV

a cassettes with various promoters

b cassettes with virus-derived genetic modulators

c cassettes with hybrid promoter

Figure 2.1 Transfer plasmids with different transgene expression

cassette for the production of baculovirus vectors

Baculoviral vectors carrying the above expression cassettes were

produced and propagated in Sf9 insect cells according to the Bac-to-Bac

Baculovirus Expression System manual (Invitrogen) Budded viruses in the insect cell culture medium were filtered through 0.2 μm pore size filters (Millipore, Billerica, MA, http://www.millipore.com) to remove any cell debris, and concentrated by centrifugation at 28,000 g for 60 min Viral pellets were re-suspended in 0.1 M phosphate-buffered saline (PBS) and their infectious titers (plaque-forming units, pfu) were determined using a plaque assay on

Sf9 cells

2.2.2 Maintenance of hES cells and Baculoviral Transduction

Two hES cell lines HES-1 (NIH code: ES01) and HES-3 (NIH code: ES03)(Reubinoff et al., 2000), both of which are listed on National Institutes

of Health (NIH) Human Embryonic Stem Cell Registry, were used These two hES cell lines and their feeder cells K4 mouse embryonic fibroblasts (mEFs) were obtained from ES Cell International (ESI Singapore, http://www.escellinternational.com) A protocol provided by ESI was used to amplify and maintain these two cell lines Both HES-1 and HES-3 cells used for baculoviral transduction experiments were further amplified on mitotically inactivated mEFs (CF-1 from American Type Culture Collection, Manassas,

VA, http://www.atcc.org) seeded in gelatin (Sigma-Aldrich, St Louis, http://www.sigmaaldrich.com) -coated dishes in 80% knock-out DMEM (Invitrogen) supplemented with 20% knockout serum replacement

(Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino

acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 4 ng/mL basic

fibroblast growth factor (Invitrogen), 50 U/mL penicillin, 50

µg/mLstreptomycin as described before(Zeng et al., 2007) The hES colonies

were subcultured every 7 days by mechanical slicing and replating into

culture dishes with fresh mEFs

During transduction, baculoviral vectors in 100　μl PBS were added at a

desired multiplicity of infection (MOI) to a suspension of HES-1 or HES-3 cell

clumps in 50　μl knock-out DMEM After transduction for 2 h, the hES cell

clumps were washed with PBS and further cultured For routine transduction

experiments, hES cell clumps of normal size isolated at the time of

subculturing were used and replated on mEFs in the above-mentioned

medium after transduction To facilitate fluorescence-activated cell sorting

(FACS) analysis of multiple samples, the hES cell clumps were mechanically

cut to a size of about 200 cells per clump for transduction and the transduced

clumps were replated and cultured with a feeder-free method by seeding onto

Matrigel (BD Biosciences, Bedford, MA, http://www.bdbiosciences.com )-coated plates in 80% knock-out DMEM

supplemented with 20% knockout serum replacement, 2 mM L-glutamine, 0.1

mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 40 ng/mL basic

fibroblast growth factor, 50 U/mL penicillin, 50 µg/mLstreptomycin (Xu et al.,

2005)

2.2.3 FACS Analysis

Two days after transduction, the transduced hES cells were washed in PBS, trypsinized into single cells, and analyzed using a FACSCalibur flow cytometer (BD Biosciences) The use of eGFP as a reporter allows the evaluation of baculoviral transduction efficiency using both the percentage of eGFP-positive cells and the mean fluorescence intensity (MFI) of eGFP The results of the FACS analysis are given as mean ± standard deviation (SD)

from three to five experiments and were analyzed using Student’s t-test

2.2.4 Immunocytochemistry

For hES cell immunofluorescence staining, the transduced hES cells were washed with PBS and fixed with ice-cold absolute methanol for 15 min This was followed by permeabilization with PBS containing 0.1% Triton X-100 for

30 s and blocking with 1% BSA in PBS for 30 min The samples were then incubated with primary monoclonal antibodies against the hES cell marker TRA-1-60 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) for 1 h After washing, a goat anti-mouse IgM-PE (Santa Cruz Biotechnology) secondary antibody was used for localization To detect eGFP protein, Alexa Fluor 488 labeled- rabbit anti-GFP antibody (Invitrogen Molecular Probes) was included for double immunostaining The samples were counterstained with Hoechst before observation

2.3 Results

2.3.1 Optimization of Baculoviral Transduction Method

There are two commonly used transduction methods for viral transduction: adherent transduction and suspension transduction Since baculovirus vectors have been routinely applied to mammalian cells by the adherent method, we tested this method first with HES-1 cells hES cells were cultured

on mEFs and transduced with baculoviruses containing a EGFP reporter gene at desired MOI in serum-free medium at 37ºC for 1 h On day 1 after infection, mass transgene expression was observed in the feeder mEF cells

at a MOI of 50 (Figure 2.2a), suggesting that the virus preparation was of good quality On day 2, there were a few cells with detectable transgene expression in the hES colonies, representing no more than 1% of the total hES cells (Figure 2.2b) hES cells grown on feeder displayed a much lower percentage of transgene expression compared with the feeder cells, indicating the massive uptake of baculovirus by the mouse feeders

Figure 2.2 Baculovirus-mediated transgene expression in HES-1 cells grown on feeders The hES cells grown on feeders were infected with

recombinant baculovirus containing the EGFP gene under control of the CMV promoter at MOI 200 Overlaying of phase-contrast and fluorescence images (left) and fluorescence microscopy images (right) of a hES cell colony on day 1 after infection to show transduced cells On the bottom, a high magnification of the hES cell colony to show transduced hES cells in the colony

a

b

2.3.2 Baculoviral Vectors Effectively Mediate Gene Transfer to hES Cells

We isolated hES cell clumps of the HES-1 line by mechanical slicing, suspended them in serum-free hES cell culture medium and transduced the clumps with baculoviral vectors containing an eGFP gene at an MOI of 100 for 2 h before replating on feeders Using a baculoviral vector incorporating the human EF1α promoter, we observed eGFP expression as early as 6 h after transduction The expression became intense at 24 h, with bright green fluorescence observed throughout the clumps (Figure 2.3a) hES cells in the clumps maintained outgrowth after transduction, resulting in colonies with a central ‘green’ region surrounded by newly generated, eGFP-negative hES cells (Figure 2.3a, Day 4) This finding suggests that the virus was unable to replicate and to deliver its genome to hES cell progeny The eGFP signals in the central region decreased over time (Figure 2.3a, Day 1-6) and became very weak after day 6 Using baculoviral vectors with both the EF1α promoter and 　 the WPRE, up to 80% of the cells in the infected hES cell clumps were eGFP-positive at day 2 as analyzed by flow cytometry (Figure 2.3b); while using baculoviral vectors with the CMV promoter or the human EF1α promoter without the WPRE, the transduction efficiency was approximately 25 to 40% (Figure 2.3b) The baculoviral vectors were also able to transduce embryoid bodies derived from hES cells (Figure 2.3c) We conclude that baculoviral vectors are effective in transducing not only