vii
TABLE OF CONTENTS
CHAPTER
TITLE
PAGE
TITLE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xiv
CHAPTER 1 INTRODUCTION
1
1.1
Introduction
1
1.2
Research Background
2
1.3
Problem Statement
5
viii
1.4
Objectives
6
1.5
Scope of Study
6
1.6
Significant of Study
7
CHAPTER 2 LITERATURE REVIEW
8
1.2
Introduction
8
2.2
Gene delivery
8
2.3
Gene therapy
9
2.4
Vectors and Methods for Gene Therapy
10
2.4.1 Viral Vectors
11
2.4.2 Physical methods
14
2.4.2.1
Needle Injection of Naked DNA
14
2.4.2.2
Gene Gun or Ballistic DNA Injection
15
2.4.2.3
Gene Electroporation
15
2.4.3 Non-viral Vectors
17
2.4.3.1
Cationic polymers
18
2.4.3.2
Cationic lipids
18
2.4.3.3
Hybrid of lipid and polymer
19
2.4.3.4
Porphyrins
20
i.
Porphyrin Structure and Roles
20
ii.
Porphyrin Interaction with DNA
21
2.5
Chinese Hamster Ovarian (CHO) Cells
24
2.6
MTT Assay
25
2.7
Enhanced Green Fluorescent Protein (EGFP)
26
CHAPTER 3 MATERIAL AND METHODS
3.1
28
Materials
28
3.1.1 Chemicals and Reagents
28
3.1.2 Solutions and Buffers
29
3.1.2.1 Bacterial Growth Media
29
3.1.2.2 Cell Line Complete Growth Media
29
ix
3.1.2.3 MTT Assay’s Solutions and Buffers
3.2
30
Methodology
30
3.2.1 Cell Culture Techniques
30
3.2.1.1 Chinese Hamster Ovary (CHO) Cells
Recovery
30
3.2.1.2 Chinese Hamster Ovary (CHO) Cells
Subculture
3.2.2 Cell Viability Determination by MTT Assay
3.2.2.1 MTT Assay Procedure
31
32
34
3.3
Culturing Transformed Cells
35
3.4
Plasmid Extraction (Mini Prep)
36
3.4.1 Plasmid Identification and Purification Procedure
37
3.4.2 Determination of Plasmid DNA Purification and
3.5
Concentration
38
Gene Transfection
38
3.5.1 Counting Cell by Using Haemocytometer
39
3.5.2 Gene Transfection Procedure for X-treme DNA
HP
40
3.5.3 Gene Transfection Procedure for Porphyrins’
3.6
Derivatives
41
3.5.4 Fluorescent Analytical Method
42
Statistical Analysis
42
CHAPTER 4 RESULTS AND DISCUSSION
43
4.1
Introduction
43
4.2
Chinese Hamster Ovary cells
43
4.3
Evaluation of MTT Assay
44
4.4
Culturing Transformed Cell
50
4.5
Extraction and Purification of Plasmid
51
4.6
Determination of Plasmid DNA Purification and
Concentration
51
x
4.7
Cellular Uptake and Gene Transfection
CHAPTER 5 CONCLUSION AND RECOMMENDATIONS
53
61
5.1
Conclusion
61
5.2
Recommendations
62
REFERENCES
63
APPENDICES
73
Appendix A
73
xi
LIST OF TABLES
TABLE NO.
TITLE
PAGE
2.1
Classifications of viral vectors for gene delivery
13
2.2
Physical methods of gene transfection.
16
3.1
Design of MTT assay
33
4.1
OD reading of miniprep plasmid using Nanodrop ND-1000
spectrophotometer
51
xii
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
Cationic porphyrin derivatives’ structures
7
2.1
Basic structure of porphyrin
21
2.2
Non-viral gene delivery by using cationic compounds
22
2.3
Cleavage of MTT to its formazon by mitochondrial reductase
enzyme
25
2.4
Physical map of pIRES2-EGFP
27
3.1
Design of MTT assay on 96-well microplate
32
3.2
General flow chart for MTT assay
34
3.3
Pattern for counting cells using haemocytomete
40
3.4
Gene transfection procedure
41
4.1
Cell growth pattern
44
4.2
Result of MTT assay on CHO cells
46
4.3
Cell viability percentages versus DMSO5% and different
concentrations of Porphyrin derivatives
49
xiii
4.4
Growth of E.coli DH5α containing the plasmid DNA on solid
agars(a) and liquid media(b)
4.5
Example of OD reading of miniprep plasmid using Nanodrop
ND-1000 spectrophotometer
4.6
50
52
Cellular uptake and gene transfection results of Porphyrin
derivatives at different concentrations compared with
positive control
58
xiv
LIST OF ABBREVIATIONS
µg
-
Microgram
µM
-
Micro Molar
µL
-
Micro Litter
ANOVA
-
Analysis Of Variance
cDNA
-
complementary deoxyribonucleic acid
CHO
-
Chinese Hamster Ovary
CO2
-
carbon dioxide gas
CTAB
-
cetyltrimethylammonium bromide
dH2O
-
distilled water
DMSO
-
Dimethyl sulfoxide
DMEM
-
Dulbecco’s Modified Eagle Medium
DNA
-
deoxyribonucleic acid
DNase
-
deoxyribonuclease
EGFP
-
Enhanced green Fluorescent protein
ELISA
-
Enzyme-linked immunosorbent assay
FBS
-
fetal bovine serum
G
-
Gram
GFP
-
Green fluorescent protein
HCl
-
hydrochloric acid
LB
-
Luria Broth
min
-
minute(s)
ml
-
Mili Litter
xv
MTT assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
MW
-
molecular weight
NaCl
-
sodium chloride
NaOH
-
sodium hydroxide
nm
-
nanometer
Pcmv
-
Cephalomyocarditis virus plasmid
PBS
-
Phosphate Buffer Saline
PEI
-
Polyetheleneamine
PAMAM
-
polyamidoamine G4-porphyrin conjugate
Pophyrin.D1 -
5-hexyl-10,15,20tris(N-methyl-4-pyridyl)porphyrin
Pophyrin.D2 -
5-propyl-10,15,20tris(N-methyl-4-pyridyl)porphyrin
Pophyrin.D3 -
5,10-dipropyl-15,20-bis (N-methyl-4-pyridyl)porphyrin,
Pophyrin.D4 -
5,10-dihexyl-15,20bis(N-methyl-4-pyridyl)porphyrin
RNA
-
ribonucleic acid
RNase
-
ribonuclease
rpm
-
round per minute
SDS
-
Sodium dodecyl sulfate
Sec
-
second(s)
UV
-
Ultraviolet