Table of Contents
Preface xiv
Acknowledgments xv
Author biographies xvi
1 History, scope and development of biotechnology 1-1
1.1 Introduction 1-1
1.2 Branches of biotechnology 1-5
1.3 Biotechnology and its various stages of development 1-6
1.3.1 Old and new biotechnology 1-17
1.3.2 Ancient biotechnology (pre-1800) 1-17
1.3.3 Classical biotechnology 1-19
1.3.4 Modern biotechnology 1-21
1.4 Scope and importance of biotechnology 1-27
1.4.1 Biotechnology in medicine 1-27
1.4.2 Industrial biotechnology 1-28
1.4.3 Biotechnology and the environment 1-29
1.4.4 Biotechnology and agriculture 1-29
1.5 Biotechnology techniques 1-29
1.5.1 Bioreactors 1-29
1.5.2 Cell fusion 1-29
1.5.3 Liposome-based delivery 1-31
1.5.4 Cell or tissue culture 1-32
1.5.5 Genetic engineering 1-32
1.5.6 DNA fingerprinting 1-32
1.5.7 Cloning 1-32
1.5.8 Artificial insemination and ET technology 1-33
1.5.9 Stem cell technology 1-33
1.6 Applications of biotechnology 1-34
1.6.1 Basic applications of biotechnology 1-35
1.6.2 Most common applications 1-35
1.7 Biotech research: 2015-2016 1-47
References 1-57
2 Modern DNA science and its applications 2-1
2.1 Introduction 2-1
2.1.1 Genes: units of inheritance 2-1
2.2 The Human Genome Project 2-3
2.3 DNA synthesis begins at replication origins 2-4
2.4 Gene expression 2-4
2.4.1 One gene, one product 2-4
2.5 Structure of DNA 2-6
2.5.1 Basic structural features of DNA 2-7
2.6 DNA replication 2-7
2.6.1 Leading strand 2-8
2.6.2 Lagging strand 2-9
2.7 DNA supercoiling 2-9
2.8 Repair and recombination 2-9
2.9 Types of DNA damage 2-11
2.9.1 Base excision repair (BER) 2-13
2.9.2 Mismatch repair (MMR) 2-13
2.9.3 Nucleotide excision repair (NER) 2-14
2.9.4 Double-strand break (DSB) repair 2-14
2.10 DNA recombination 2-14
2.10.1 Types and examples of recombination 2-15
2.11 DNA isolation is the commencement of molecular marker analysis 2-16
2.11.1 DNA extraction protocols 2-17
2.12 Types of molecular markers 2-18
2.12.1 Hybridization-based molecular markers 2-18
2.12.2 PGR-based markers 2-22
2.13 Genomic library screening methods 2-34
2.13.1 Colony hybridization 2-34
2.13.2 Chromosome walking 2-35
2.13.3 Blotting techniques 2-36
2.13.4 Southern blot analysis 2-36
2.13.5 Northern blot analysis 2-36
2.13.6 Western blot analysis 2-37
2.13.7 Dot blot technique 2-37
2.13.8 Techniques for the detection of specific proteins 2-38
2.13.9 Electrophoresis techniques 2-38
2.14 Triplex DNA, TFOS, PNAs, RNA-DNA hybrids and DSRNA/RNAI 2-44
2.15 Isolation, sequencing and synthesis of genes 2-46
2.15.1 Isolation of ribosomal RNA 2-46
2.15.2 Isolation of genes coding for specific proteins 2-46
2.15.3 Isolation of genes (with known or unknown products) using DNA or RNA probes 2-47
2.15.4 Isolation of genes (with known or unknown products) using DNA or RNA probes 2-48
2.15.5 Use of transposable elements (transposon tagging) 2-49
2.15.6 T-DNA insertion mutagenesis for isolation of plant genes 2-50
2.15.7 Promoter, enhancer and gene trap for isolation of genes 2-50
2.15.8 Mutation complementation 2-51
2.15.9 Differential screening and differential display technique for isolation of genes 2-51
2.15.10 Subtractive hybridization for gene isolation 2-52
2.15.11 Map-based cloning for gene isolation 2-52
2.15.12 Isolation of novel genes 2-52
2.15.13 Sequencing of a gene or a DNA fragment 2-53
2.16 Synthesis of genes 2-54
2.16.1 Chemical synthesis of tRNA genes 2-54
2.16.2 Synthesis of the gene for yeast alanyl tRNA 2-56
2.16.3 Synthesis of a gene from true precursor tRNA 2-56
2.16.4 Mass spectrometry for genomics and proteomics 2-56
2.17 Genomics and proteomics research 2-60
References 2-61
3 Introduction to genetic engineering 3-1
3.1 Introduction 3-1
3.2 Gene transfer technologies 3-2
3.2.1 Electro poration 3-2
3.2.2 Microinjection 3-3
3.2.3 Biolistics or microprojectiles for DNA transfer 3-3
3.2.4 Liposome-mediated gene transfer 3-4
3.2.5 Calcium-phosphate-mediated DNA transfer 3-4
3.2.6 DNA transfer by DEAE-dextran method 3-5
3.2.7 Transfer of DNA by polycation-DMSO 3-6
3.2.8 Poly ethylene-gly col-mediated transfection 3-7
3.2.9 Gene transfer through peptides 3-7
3.2.10 Gene transfer by retroviruses 3-8
3.3 Plasmids 3-9
3.4 Different hosts and protein expression technologies 3-10
3.4.1 rDNA technology 3-12
3.5 Gene cloning 3-15
3.6 Transfection methods and transgenic animals 3-16
3.6.1 Gene transfer or transfection 3-17
3.6.2 Transfection of fertilized eggs or embryos 3-17
3.6.3 Transfer of whole nuclei (or split embryos) 3-17
3.6.4 DNA microinjection into the egg 3-18
3.6.5 Virus-mediated gene transfer to embryo 3-19
3.6.6 Transfection of cultured mammalian cells 3-20
3.6.7 Targeted gene transfer 3-21
3.6.8 Transgenic animals in biotechnology 3-21
3.7 Applications of genetic engineering in biotechnology 3-30
3.8 Mammalian cell line characterization 3-32
3.9 In vitro fertilization (IVF) and embryo transfer in humans and domestic animals 3-33
3.10 IVF in humans and embryo transfer 3-34
3.10.1 Types and causes of infertility 3-34
3.10.2 Evaluation and assessment of patients 3-37
3.10.3 IVF fertility treatment 3-38
3.10.4 Development of ovarian follicles in natural menstrual cycles 3-39
3.10.5 Development of ovarian follicles in stimulated cycles 3-39
3.10.6 Development of ovarian follicles during a controlled cycle 3-40
3.10.7 Ovarian stimulation protocols for IVF 3-40
3.10.8 Spontaneous luteinizing hormone (LH) surge 3-43
3.10.9 Administration of hCG for controlled ovulation 3-43
3.10.10 Equipment and technique for laparoscopy 3-44
3.10.11 Oocyte culture and IVF culture of oocytes 3-45
3.10.12 Preparation of semen 3-46
3.10.13 In vitro fertilization 3-47
3.11 Embryo transfer (ET) in humans 3-48
3.11.1 Time of ET 3-48
3.31.1 ET technique 3-48
3.12 Superovulation, IVF and embryo culture in farm animals 3-50
3.13 ET in cattle 3-52
3.13.1 ET technique in cattle 3-52
3.13.2 Technique for freezing embryos in cattle 3-54
3.13.3 Benefits of ET in cattle 3-55
References 3-55
4 Applications of stem cells in disease and gene therapy 4-1
1.1 Introduction
4.2 Types of gene therapy 4-2
4.2.1 Somatic gene therapy 4-2
4.2.2 Gennline gene therapy 4-3
4.3 Gene therapy strategies 4-3
4.3.1 Gene augmentation therapy (GAT) 4-3
4.3.2 Targeted killing of specific cells 4-4
4.3.3 Targeted inhibition of gene expression 4-5
4.3.4 Targeted gene mutation correction 4-5
4.4 Methods of gene therapy 4-5
4.1.1 Ex vivo gene therapy 4-6
4.1.2 In vivo gene therapy 4-7
4.5 Gene therapy approaches 4-8
4.5.1 Conventional gene therapy 4-10
4.5.2 Non-classical gene therapy 4-10
4.6 Vectors for gene therapy 4-10
4.6.1 Viral-mediated gene delivery 4-10
4.6.2 Non-viral-mediated gene therapy 4-11
4.7 Target sites for gene therapy 4-19
4.7.1 Target cells for gene transfer 4-19
4.8 Gene therapy strategies for cancer 4-20
4.8.1 Tumor necrosis factor gene therapy 4-20
4.8.2 Suicide gene therapy 4-20
4.8.3 Two gene cancer therapy 4-21
4.8.4 Gene replacement therapy 4-21
4.9 Gene therapy for AIDS 4-22
4.9.1 REV and ENV genes 4-22
4.9.2 Genes of HIV proteins 4-22
4.9.3 Gene to inactivate gpl20 4-22
4.10 Oligonucleotide therapies: anttgene and antisense therapy 4-22
4.10.1 Antisense therapy for cancer 4-23
4.10.2 Antisense therapy for AIDS 4-25
4.11 Antisense oligonucleotides as therapeutic agents 4-26
4.12 Chimeric oligonucleotides in gene correction 4-27
4.13 Aptamers as therapeutic agents 4-28
4.14 Ribozymes as therapeutic agents 4-29
4.15 The future of gene therapy 4-29
4.16 Stem cell research 4-30
4.16.1 Stem cell classification 4-30
4.16.2 Historical background of stem cell research 4-32
4.16.3 Ethical issues associated with cell lines 4-33
4.16.4 Applications of stem cell research 4-33
4.16.5 Human ES ceils 4-34
References 4-35
5 Transgenic animals in biotechnology 5-1
5.1 Introduction 5-1
5.2 Major objectives of gene transfer 5-2
5.3 Cloning vectors 5-2
5.3.1 Fish vectors in molecular genetics and biotechnology 5-5
5.3.2 P element vectors/transposon 5-5
5.3.3 Baculovirus as versatile vectors for protein expression in insects 5-7
5.4 Efficient and versatile mammalian virus vectors 5-8
5.4.1 SV40 vectors 5-8
5.4.2 BPV (bovine papillomavirus) DNA vectors 5-14
5.4.3 Retrovirus vectors and their use 5-14
5.4.4 Vaccinia virus vectors: new approach for producing recombinant vaccines 5-16
5.4.5 Adenovirus vectors (for gene therapy, vaccination and cancer gene therapy) 5-16
5.5 Mammalian artificial chromosome (MAC) vectors for somatic gene therapy 5-17
5.6 DNA constructs 5-18
5.6.1 Promoter sequences 5-19
5.6.2 Selectable reporter or marker genes 5-19
5.7 Transfection methods: an approach towards mammalian cell transfection 5-24
5.7.1 Calcium phosphate-mediated transfection of eukaryotic cells 5-24
5.7.2 Transfection using DEAE-dextran 5-25
5.7.3 Lipofection (lipid-mediated DNA transfection method) 5-26
5.7.4 Bacterial protoplast fusion 5-28
5.7.5 Gene transfer by electroporation 5-28
5.7.6 Retrovirus-mediated gene transfer 5-30
5.7.7 Basics of DNA microinjection 5-31
5.8 Xenopus oocytes as a heterologous expression system 5-34
5.9 ES cell-mediated gene transfer 5-35
5.10 Targeted gene transfer or gene therapy in mammals 5-37
5.10.1 Gene disruption by. HR in mammals 5-39
5.10.2 Gene targeting/replacement 5-40
5.11 Transgene integration, organization and expression 5-45
5.12 Transgene recovery in mammahan cells 5-46
5.13 Cloned protein expression in mammalian cells 5-48
5.13.1 Expression vectors for mammalian cells 5-50
5.13.2 Enhanced production of recombinant proteins 5-56
5.13.3 Scale-up of protein purification (stages in downstream processing) 5-60
References 5-63
